Vaccines are biological preparations that provides active acquired immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins, or one of its surface proteins. The agent stimulates the body's immune system to recognize the agent as a threat, destroy it, and to further recognize and destroy any of the microorganisms associated with that agent that it may encounter in the future. In the 2000s, vaccine misinformation developed, with opponents of vaccination spreading false claims through various media.
- Most of the objections made by doctors with occult tendencies are based unconsciously on a feeling that there should be higher methods of controlling diseases in man than by injecting into the human body substance taken from the bodies of animals. That is most surely and definitely correct, and some day it will be demonstrated. ... A more vital objection should be based on the suffering entailed on the animals providing the vaccine and other substances...the effect on the inner bodies is practically nil, and far less than the diseases themselves...The controlling of modern disease is being handled by modern medicine primarily in three ways: through the science of sanitation, through preventive medicine, and through inoculation.
- Alice Bailey in Esoteric Healing (A Treatise on the Seven Rays), p.322/4, (1953).
- The science of inoculation is purely physical in origin, and concerns only the animal body. This latter science will shortly be superseded by a higher technique, but the time is not yet.
- Alice Bailey in Esoteric Healing (A Treatise on the Seven Rays) p.322/4 (1953).
- How does sleep affect immunity during a genuinely ongoing immune response? There are quite a few studies that investigated the effects of sleep on the response to vaccinations used as an experimental model of infection. Intriguingly, these studies consistently demonstrate that sleep enhances the adaptive immune response against the invading antigen. Compared with subjects who stayed awake during the night after a single vaccination against hepatitis A in the morning before, subjects who regularly slept on this first night after vaccination, 4 weeks later, displayed a twofold increase in antigen-specific antibody titres. This study was the first to show in humans that a single night of normal sleep after vaccination strengthens the evolvement of a natural immune response against an invading antigen, to a clinically relevant extent. Subsequent experiments confirmed these effects for repeated inoculations with both hepatitis A and B antigens and showed that the immune-boosting effect of nocturnal sleep was also reflected by a doubling of the number of circulating antigen-specific Th cells that drive the production of hepatitis A and B-specific antibodies (Fig. 4a). The proportion of pro-inflammatory and Th1 cytokine (IL-2, IFN-γ, TNF-α) producing T cells was also profoundly reinforced by sleep. Importantly, these immuno-enhancing effects of sleep were still present at a 1-year follow-up, indicating that sleep in enhancing the initial formation of an adaptive immune response also supports the long-term maintenance of the antigenic memory, a function hallmarking the immune system.
- Luciana Besedovsky, Tanja Lange,and Jan Born; “Sleep and immune function”, Pflugers Arch. 2012 Jan; 463(1): 121–137.
- Edward R. Murrow: Who owns the patent on this vaccine?
Jonas Salk: Well, the people, I would say. There is no patent. Could you patent the sun?
- Although the time periods have changed, the emotions and deep-rooted beliefs—whether philosophical, political, or spiritual—that underlie vaccine opposition have remained relatively consistent since Edward Jenner introduced vaccination.
- College of Physicians of Philadelphia, The History of Vaccines.
- For scientific research and for the production of vaccines or other products, cell lines are at times used which are the result of an illicit intervention against the life or physical integrity of a human being. The connection to the unjust act may be either mediate or immediate, since it is generally a question of cells which reproduce easily and abundantly. This "material" is sometimes made available commercially or distributed freely to research centers by governmental agencies having this function under the law. All of this gives rise to various ethical problems with regard to cooperation in evil and with regard to scandal. It is fitting therefore to formulate general principles on the basis of which people of good conscience can evaluate and resolve situations in which they may possibly be involved on account of their professional activity.
It needs to be remembered above all that the category of abortion "is to be applied also to the recent forms of intervention on human embryos which, although carried out for purposes legitimate in themselves, inevitably involve the killing of those embryos. This is the case with experimentation on embryos, which is becoming increasingly widespread in the field of biomedical research and is legally permitted in some countries... [T]he use of human embryos or fetuses as an object of experimentation constitutes a crime against their dignity as human beings who have a right to the same respect owed to a child once born, just as to every person". These forms of experimentation always constitute a grave moral disorder.
- Congregation for the Doctrine of the Faith, "Dignitas Personae: On Certain Bioethical Questions”, “Third Part: New Problems Concerning Procreation”, “The use of human "biological material" of illicit origin”, n.34, (20 June 2008).
- Grave reasons may be morally proportionate to justify the use of such "biological material". Thus, for example, danger to the health of children could permit parents to use a vaccine which was developed using cell lines of illicit origin, while keeping in mind that everyone has the duty to make known their disagreement and to ask that their healthcare system make other types of vaccines available. Moreover, in organizations where cell lines of illicit origin are being utilized, the responsibility of those who make the decision to use them is not the same as that of those who have no voice in such a decision.
In the context of the urgent need to mobilize consciences in favour of life, people in the field of healthcare need to be reminded that "their responsibility today is greatly increased. Its deepest inspiration and strongest support lie in the intrinsic and undeniable ethical dimension of the health-care profession, something already recognized by the ancient and still relevant Hippocratic Oath, which requires every doctor to commit himself to absolute respect for human life and its sacredness".
- Congregation for the Doctrine of the Faith, "Dignitas Personae: On Certain Bioethical Questions”, “Third Part: New Problems Concerning Procreation”, “The use of human "biological material" of illicit origin”, n.35, (20 June 2008).
- “There is an expression out there that a failed gene therapy makes a good vaccine,” says Luk Vandenberghe, a viral vector expert at Harvard Medical School.
One attractive feature is that adenoviruses’ inflammatory effects mean developers don’t have to use adjuvants, molecules added to conventional vaccines to direct the immune system’s attention to the viral protein. The adenoviruses themselves drive the inflammation, which is kept under control by giving the vaccines at low doses.
And all genetic vaccines—DNA vaccines, mRNA vaccines, and adenoviral vector vaccines—mimic a natural viral infection by forcing our bodies to produce viral proteins inside our cells. That spurs the T cells of our immune system to attack these vaccinated cells, and in the process, they learn to seek and destroy cells infected with the real virus in the future.
Traditional vaccines, made from weakened viruses or viral proteins, stimulate B cells to make antibodies against the virus. Those antibodies latch onto invading viruses and prevent them from entering our cells.
The problem is that once the virus infiltrates our cells, the antibodies from a traditional vaccine are useless. It’s at that stage that T cells need to swoop in. Adenovirus vectors “are the best of all vaccines at inducing a T-cell response,” Wistar’s Ertl says.
- Ryan Cross, “Adenoviral vectors are the new COVID-19 vaccine front-runners. Can they overcome their checkered past?”, Vol. 98, Issue 12, (May 12, 2020)
- Vaccines, even in their present rather coarse form, have been of immense benefit to humanity and have all but eradicated many formerly killer diseases. Especially in (homeopathically) refined form they will have a long-term value in dealing with disease.
- Benjamin Creme, Maitreya’s Mission, Volume II, p. 511 (1993)
- While vaccines (excepting those homoeopathically produced) may not be the best and final solution to conquering diseases, on a mass scale they have brought much benefit and freedom from crippling diseases in Africa, India and many other countries.
- Benjamin Creme in Share International magazine, (May 2002).
- Petri described the new study as paradigm-shifting. “It was one of those rare, seminal findings that changes the way I think about the immune response,” he said.
Davis’ study offers hope that some of the immunity conferred by a vaccine extends beyond the specific microbe it targets, Petri said. “This adds support to the impetus to vaccinate infants in the developing world,” he said. As many as 30 different pathogens can cause diarrhea, so vaccinating small children against all of them — even if those vaccines existed — would require so many separate injections as to be logistically hopeless. Understanding the mechanism by which cross-reactivity occurs might further allow immunologists to develop “wide-spectrum vaccines” that cover a number of infectious organisms.
- Bruce Goldman, “Immune systems of healthy adults 'remember' germs to which they’ve never been exposed, Stanford study finds”, “Stanford Medicine”, (Feb. 7, 2013)
- A vaccine is just exposing yourself to a little bit of the bad thing that can kill you.
- Sarah Gonzalez, “The Very First Vaccine”, Planet Money, NPR, (June 10, 2020)
- Several accounts from the 1500s describe smallpox inoculation as practices in China and India (one is referred to in volume 6 of Joseph Needham’s Science and civilization in China). Glynn and Glynn, in the The Life and Death of Smallpox, note that in the late 1600s Emperor K’ang His, who had survived smallpox as a child, had his children inoculated. That method involved grinding up smallpox scabs and blowing the matter into nostril, inoculation may also have been practiced by scratching matter from a smallpox sore into the skin. It is difficult to point when the practice began, as some sources claim dates as early as 300 BCE.
- History of vaccines.org, Chinese Smallpox Inoculation, College of Physicians of Philadelphia.
- I am not surprised that men are not thankful to me; but I wonder that they are not grateful to God for the good which he has made me the instrument of conveying to my fellow-creatures.
- The history of medical research and human experimentation reveals both great successes and horrible abuses. Plagues like smallpox were rampant and capable of wiping out entire cities. People were desperate for relief and would try anything that could help ward off the horrible plagues, even experimenting. English aristocrat Lady Mary Wortley Montague introduced the idea of variolation to the gentry in 1715. In variolation, ooze from the sores of smallpox victims with mild cases was scratched into the skin. During the French the Indian War, General George Washington was convinced that his most formidable for was smallpox and he subjected his men to forced variolation to stop its spread. Many of the soldiers had only mil reactions, but some became seriously ill and died. The European press, especially among the antivaccine society, bitterly criticized Washington for forcing his men into possible harm without their consent, Hessian soldiers, who fought alongside the British, were captured and imprisoned in Frederick, Maryland, where they may have been subjected to variolation experimentation—a safety precaution before Washington would order to the procedure for his own army. When British physician Edward Jenner (1749-1823) introduced the use of cowpox sores to make a vaccine against smallpox, he was subjected to the same criticism.
In the 1700s principles of individualism, self-determination, and consent of the governed formed the establishment of the United States. Ethicists all this idea the principle of “respect for persons.” Therefore, informed consent is a human right and an outgrowth of life, liberty and the pursuit of happiness.
- During the French Indian War, General George Washington was convinced that his most formidable for was smallpox and he subjected his men to forced variolation to stop its spread. Many of the soldiers had only mild reactions, but some became seriously ill and died. The European press, especially among the antivaccine society, bitterly criticized Washington for forcing his men into possible harm without their consent, Hessian soldiers, who fought alongside the British, were captured and imprisoned in Frederick, Maryland, where they may have been subjected to variolation experimentation-a safety precaution before Washington would order to the procedure for his own army.
- There is no longer any reason why American children should suffer from polio, diphtheria, whooping cough, or tetanus—diseases which can cause death or serious consequences throughout a lifetime, which can be prevented, but which still prevail in too many cases.
I am asking the American people to join in a nationwide vaccination program to stamp out these four diseases, encouraging all communities to immunize both children and adults, keep them immunized, and plan for the routine immunization of children yet to be born. ...[O]ver the next 3 years, I am proposing legislation authorizing a program of federal assistance. This program would cover the full cost of vaccines for all children under five... It would also assist in meeting the cost of organizing the vaccination drives... and the cost of extra personnel... [T]he legislation provides continuing authority to permit a similar attack on other infectious diseases... susceptible of practical eradication as a result of new vaccines or other preventive agents. Success in this effort will require the whole-hearted assistance of the medical and public health professions, and a sustained nationwide health education effort.
- John F. Kennedy, "Special Message to the Congress on National Health Needs" (February 27, 1962) To the Congress of the United States.
- The history of medical research and human experimentation reveals both great successes and horrible abuses. Plagues like smallpox were rampant and capable of wiping out entire cities. People were desperate for relief and would try anything that could help ward off the horrible plagues, even experimenting. English aristocrat Lady Mary Wortley Montague introduced the idea of variolation to the gentry in 1715. In variolation, ooze from the sores of smallpox victims with mild cases was scratched into the skin. During the French and Indian War, General George Washington was convinced that his most formidable for was smallpox and he subjected his men to forced variolation to stop its spread. Many of the soldiers had only mild reactions, but some became seriously ill and died. The European press, especially among the antivaccine society, bitterly criticized Washington for forcing his men into possible harm without their consent, Hessian soldiers, who fought alongside the British, were captured and imprisoned in Frederick, Maryland, where they may have been subjected to variolation experimentation-a safety precaution before Washington would order to the procedure for his own army. When British physician Edward Jenner (1749-1823) introduced the use of cowpox sores to make a vaccine against smallpox, he was subjected to the same criticism.
In the 1700s principles of individualism, self-determination, and consent of the governed formed the establishment of the United States. Ethicists all this idea the principle of “respect for persons.” Therefore, informed consent is a human right and an outgrowth of life, liberty and the pursuit of happiness.
- Even if antibody responses seem robust, that could be misleading, cautions Melinda Beck, who studies the relationship between nutrition and immune responses at the University of North Carolina. In her studies, she says, obese people have normal initial antibody levels in response to flu vaccines, but are still twice as likely as vaccinated lean people to get the flu (that’s not to say that vaccination offers obese people no benefit). And analyses so far have focused on Western definitions of obesity. These are based on BMI, a crude measure that fails to distinguish between fat that accumulates under the skin, and fat that accumulates around organs, called visceral fat, which is more closely associated with diseases such as diabetes and high blood pressure.
In people of European descent, a BMI of 30 kilograms per square metre or above is considered obese. But Popkin notes that people in some countries in Asia, the Middle East and Latin America, for example, tend to accumulate visceral fat at lower BMIs. China is the only country to set a lower threshold — a BMI of 28 kg m–2 — for obesity, but even then, Popkin says, some Chinese researchers will report their data using Western definitions of BMI to improve their chances of publishing.
- Heidi Ledford, “How obesity could create problems for a COVID vaccine” Nature 586, (20 October 2020), 488-489.
- A team led by vaccinologist Ofer Levy at Boston Children’s Hospital in Massachusetts is working on a COVID-19 vaccine specifically for older adults, using an in-vitro screening system to identify the best adjuvants. “Vaccines were typically developed as one-size-fits-all,” he says. But a lot of features — age, sex, and even the season — affect vaccine responses, Levy says. The best combinations of adjuvant and vaccine they find will be tested in mice and then in humans.
But, in general, developing medications to improve immune function seems like a much smarter strategy than creating vaccines specifically for elderly people, says Claire Chougnet, an immunologist at Cincinnati Children’s Hospital Medical Center in Ohio, who is studying inflammation in aged mice. Vaccine development is costly and time-intensive. “In the case of an emerging virus, when you want a quick response, that makes things even more complicated if you have to do two types of vaccine,” she says. Plus, individual vaccines target specific pathogens, but an immune-boosting medication could be used with any vaccine. “That could work for flu, that could work for COVID-19. That would work for COVID-25,” she says. The approach is “extremely versatile”.
- Ofer Levy as quoted by Cassandra Willyard in “How anti-ageing drugs could boost COVID vaccines in older people”, Nature 586, (14 October 2020), pp.352-354
- Vaccines made from mRNA can be made much faster than older vaccines could, explains Margaret Liu, a vaccine researcher who chairs the board of the International Society for Vaccines and specializes in genetic vaccines. The problem, says Liu, is that mRNA is "really easily destroyed, and that's because there are many, many enzymes that will just break it apart."
- Margaret Liu as quoted by Selena Simmons-Duffins, “Why Does Pfizer's COVID-19 Vaccine Need To Be Kept Colder Than Antarctica?”, NPR, (November 17, 2020)
- Vaccinations are a cornerstone of pediatric care. Traveling children need special attention to their vaccine status. Updating all routine vaccinations and accelerating those in the primary series should be done if possible. The immune system of infants and children responds differently to different types of vaccines. Safety and efficacy considerations guide vaccine recommendations for all pediatric vaccinations. Age limitations on vaccinations may be due to safety concerns (e.g., yellow fever vaccine), immune system response capability (polysaccharide vaccines), maternal antibody interference (e.g., measles, hepatitis A), or lack of data. Travel-specific recommendations may differ for vaccinating children compared with adults based on these details.
- Sheila M. Mackell, Mike Starr, “Pediatric Travel Vaccinations”, in Travel Medicine (Fourth Edition), (2019)
- Vaccination is a clinical application of immunization designed to artificially help the body to defend itself. A vaccine against infection is a modified form of a natural immunogen, which may be either the whole pathogen, one of its components, or a toxin. A vaccine does not cause disease when administered but induces the healthy host (the vaccinee) to mount a primary response against epitopes of the modified immunogen and to generate large numbers of memory B and T cells. In an unvaccinated individual (Fig. 14-1, left panel), naïve B and T cells capable of combatting an infecting pathogen are present in relatively low numbers when the pathogen is first encountered. A primary immune response is all that can be mounted so that, in many cases, the individual becomes sick until antibodies and/or effector T cells can act to clear the attacker. In a vaccinated individual (Fig. 14-1, right panel), a collection of circulating antibodies and an expanded army of pathogen-specific memory B and T cells have already been generated prior to a first exposure to the natural pathogen. When the natural pathogen attacks, the circulating antibodies provide a degree of immediate protection from the invader. In addition, the memory B and T cells are quickly activated, and a secondary response is mounted that rapidly clears the infection before it can cause serious illness. This type of vaccination is called prophylactic vaccination because it is intended to prevent disease.
- Tak W. Mak, Mary E. Saunders, Bradley D. Jett; “Primer to the Immune Response (Second Edition)”, (2014)
- NOTE: Modern air travel has made prophylactic vaccination more important than ever, because this mode of transportation has greatly facilitated the spread of contagious pathogens. For example, in 2010, an unvaccinated child who contracted measles in Europe transmitted the virus to a fellow passenger during a flight to the U.S. This passenger then attended a conference and unwittingly exposed 270 other individuals to the disease.
Vaccination can be thought of as a form of active immunization, because the individual is administered a pathogen antigen and his/her body is responsible for activating the lymphocytes and making the antibodies necessary to provide defense against future assaults. In contrast, passive immunization is the term used to describe the transfer of protective anti-bodies from an immune individual to an unimmunized individual.
- Tak W. Mak, Mary E. Saunders, Bradley D. Jett; “Primer to the Immune Response (Second Edition)”, (2014)
- Today’s best known vaccination success story is the global campaign of the World Health Organization (WHO) to eradicate smallpox. In 1967, the WHO began its coordination of 200,000 health workers who took 10 years to vaccinate the world’s population in its remotest corners. Between 1976 and 1979, only one case of smallpox was recorded, leading to the declaration in 1980 that smallpox had been officially eradicated (Plate 14-1). A similar global immunization program against rinderpest is currently pushing this pathogen toward extinction (Box 14-2).
- Vaccination as a deliberate attempt to protect humans against disease has a short history when measured against the thousands of years that humans have sought to rid themselves of plagues and pestilence. Only in the 20th century did the practice flower into the routine vaccination of large populations. Yet, despite its relative youth, the impact of vaccination on the health of the world's peoples is hard to exaggerate. With the exception of safe water, no other intervention, not even antibiotics, has had such a major effect on mortality reduction and population growth.
Since the first vaccine was introduced by Edward Jenner (Fig. 1.1) in 1798, vaccination has controlled 14 major diseases, at least in parts of the world: smallpox, diphtheria, tetanus, yellow fever, pertussis, aemophilus influenzae type b disease, poliomyelitis, measles, mumps], rubella, typhoid, rabies, rotavirus, and hepatitis B. For smallpox, the dream of eradication has been fulfilled; naturally occurring smallpox has disappeared from the world. Cases of poliomyelitis have been reduced by 99% and this disease also is targeted for eradication. Rubella and congenital rubella syndrome have been officially declared eliminated from the Americas as of 2015. Vaccinations against many other diseases have made major headway. The path to these successes is worth examining.
- Susan L. Plotkin, Stanley A. Plotkin, in Plotkin's Vaccines (Seventh Edition), ch.1, A Short History of Vaccination, (2018)
- In the 7th century, some Indian Buddhists drank snake venom in an attempt to become immune to its effect. They may have been inducing antitoxin-like immunity. In the 16th century, Brahmin Hindus in India practiced a form of variolation by introducing dried pus from smallpox pustules into the skin of a patient. Writings that cite the use of inoculation and variolation in 10th-century China–make interesting reading but apparently cannot be verified. There is, however, 18th-century documentation of Chinese variolation. The Golden Mirror of Medicine, a medical text dated 1742, listed four forms of inoculation against Smallpox practiced in China since 1695: The nose plugged with powdered scabs laid on cotton wool. Powdered scabs blown into the nose. The undergarments of an infected child put on a healthy child for several days. A piece of cotton smeared with the contents of a vesicle and stuffed into the nose. This text, endorsed by the Imperial Court, raised the status of variolation in China, which previously had been considered just a folk remedy. Another Chinese text, published a century before Jenner’s work, stated that white cow fleas were used for smallpox prevention. The fleas were ground into powder and made into pills.
- Susan L. Plotkin, Stanley A. Plotkin, in Plotkin's Vaccines (Seventh Edition), ch.1, A Short History of Vaccination, (2018)
- A hundred and twenty million cases of the most deadly, most contagious…and least excusable…disease in medicine. For smallpox can infallibly be prevented, and only a world which had forgotten Jenner could have been taken by it unaware…or a world in which the memory of Jenner’s centuries-old prophylaxis had been systematically removed.
- Frederik Pohl, Drunkard’s Walk (1960), Chapter 15 (ellipses as in the book)
- It is courage based on confidence, not daring, and it is confidence based on experience.
- In the 1970's and 1980's vaccines became, one might say, victims of their own success. They had been so effective in preventing infectious diseases that the public became much less alarmed at the threat of those diseases, and much more concerned with the risk of injury from the vaccines themselves.
- The practice of immunisation dates back hundreds of years. Buddhist monks drank snake venom to confer immunity to snake bite and variolation (smearing of a skin tear with cowpox to confer immunity to smallpox) was practiced in 17th century China. Edward Jenner is considered the founder of vaccinology in the West in 1796, after he inoculated a 13 year-old-boy with vaccinia virus (cowpox), and demonstrated immunity to smallpox.
- The Immunization Advisory Council, “A brief history of vaccination”, “Immune.org”
- In 1798, the first smallpox vaccine was developed. Over the 18th and 19th centuries, systematic implementation of mass smallpox immunisation culminated in its global eradication in 1979.
Louis Pasteur’s experiments spearheaded the development of live attenuated cholera vaccine and inactivated anthrax vaccine in humans (1897 and 1904, respectively). Plague vaccine was also invented in the late 19th Century. Between 1890 and 1950, bacterial vaccine development proliferated, including the Bacillis-Calmette-Guerin (BCG) vaccination, which is still in use today.
In 1923, Alexander Glenny perfected a method to inactivate tetanus toxin with formaldehyde. The same method was used to develop a vaccine against diphtheria in 1926. Pertussis vaccine development took considerably longer, with a whole cell vaccine first licensed for use in the US in 1948.
Viral tissue culture methods developed from 1950-1985, and led to the advent of the Salk (inactivated) polio vaccine and the Sabin (live attenuated oral) polio vaccine. Mass polio immunisation has now eradicated the disease from many regions around the world. Attenuated strains of measles, mumps and rubella were developed for inclusion in vaccines. Measles is currently the next possible target for elimination via vaccination.
Despite the evidence of health gains from immunisation programmes there has always been resistance to vaccines in some groups. The late 1970s and 1980s marked a period of increasing litigation and decreased profitability for vaccine manufacture, which led to a decline in the number of companies producing vaccines. The decline was arrested in part by the implementation of the National Vaccine Injury Compensation programme in the US in 1986. The legacy of this era lives on to the present day in supply crises and continued media efforts by a growing vociferous anti-vaccination lobby.
- The Immunization Advisory Council, “A brief history of vaccination”, “Immune.org”
- The past two decades have seen the application of molecular genetics and its increased insights into immunology, microbiology and genomics applied to vaccinology. Current successes include the development of recombinant hepatitis B vaccines, the less reactogenic acellular pertussis vaccine, and new techniques for seasonal influenza vaccine manufacture.
Molecular genetics sets the scene for a bright future for vaccinology, including the development of new vaccine delivery systems (e.g. DNA vaccines, viral vectors, plant vaccines and topical formulations), new adjuvants, the development of more effective tuberculosis vaccines, and vaccines against cytomegalovirus (CMV), herpes simplex virus (HSV), respiratory syncytial virus (RSV), staphylococcal disease, streptococcal disease, pandemic influenza, shigella, HIV and schistosomiasis among others. Therapeutic vaccines may also soon be available for allergies, autoimmune diseases and addictions.
- The Immunization Advisory Council, “A brief history of vaccination”, “Immune.org”
- It has been recognized for centuries that some diseases never reinfect a person after recovery. Smallpox was the first disease people tried to prevent by intentionally inoculating themselves with infected matter. Inoculation originated in India or China some time before 200 BC.
The concept of immunization, or how to artificially induce the body to resist infection, received a big boost in 1796, when physician Edward Jenner inoculated a young boy in England and successfully prevented him from getting smallpox. Jenner used a lancet to scratch some infected material from a woman with cowpox (similar to smallpox) under the boy’s skin.
- Lack of immunity to disease has helped to decide the fate of entire communities, from smallpox among the Indians in the New World to syphilitic soldiers in the Old. Most people have some amount of natural immunity. The human body can take care of itself in many circumstances—cuts, colds, and minor infections disappear without major upheaval. In other cases, the body has little or no naturally occurring immunity, so if you are exposed to diseases such as polio, influenza, smallpox, hepatitis, diphtheria, measles, or whooping cough, you will probably get sick with it, unless you have been immunized.
Immunization refers to the artificial creation of immunity by deliberately infecting someone so that the body learns to protect itself. An important part of the history of immunization has been determining how to get the immunizing agent into the body. The skin, which keeps germs and mischievous substances out, is also a barrier to getting medicines and vaccines into the tissue where they can work. Physicians have used varying methods to create immunity where there is none.
- Having an effective vaccine that could produce sufficient immunity was useless without being able to get it into the body in a harmless way. Edward Jenner used a lancet and scratched two lines on James Phipps’s arm. Fifty years after Jenner, the hypodermic syringe became available. In 1885, scientist Louis Pasteur used one to vaccinate a young boy who had been bitten by a mad dog and was sure to die of rabies—the boy lived, and immunization took another giant step forward.
As more immunizing agents became available, people saw the benefit of immunizing large groups, such as soldiers. During World War I, they were vaccinated against diphtheria; during World War II, typhus and tetanus.
- Hypodermic injection remains the most common method of getting through the skin. But it is not the only technology for immunization. Engineers and scientists continue to search for alternative routes into the body, such as through the moth or nose. And continuing to solve the technological problems is critical for countries in which illness and death rates are high as a result of measles, maternal tetanus, and other preventable diseases.
A successful instrument or system must get the vaccine into the body with minimal disruption, and be cost-effective for use with billions of people. And perhaps the most important problem today—preventing reuse of syringes to avoid cross-contamination—was not even imagined in the 19th century.
- World War II accelerated vaccine development. Fear of a repetition of the 1918–19 world epidemic of influenza focused urgent attention on all viral diseases, while commercial production of antibiotics taught researchers to grow viruses with less microbe contamination. Also, investigators paid closer attention to vaccine safety and effectiveness through clinical studies before release of a vaccine to the public, especially after the yellow fever vaccine apparently caused hepatitis B in many U.S. soldiers in 1942.
Polio vaccine is made from the actual virus. For both research and production, vaccine makers needed to grow large quantities of virus. Influenza virus had been grown in chicken eggs, but this method did not polio. So researchers sought other materials in which to grow poliovirus.
In 1936, Albert Sabin and Peter Olitsky at the Rockefeller Institute demonstrated that poliovirus could grow in human embryonic brain tissue, but they feared that this method might risk central nervous system damage in those who received the vaccine. The advantage of embryonic tissue, however, was that it grew quickly.
In March 1948, John Enders, Thomas Weller, and Frederick Robbins used human embryonic skin and muscle tissue, grown in a nutrient mix with antibiotics, to prove poliovirus could infect tissue other than nerve cells. Their confirmation meant that researchers could now grow enough poliovirus to create large quantities of vaccine.
The three scientists won the Nobel Prize in Physiology or Medicine in 1954, the year polio vaccine had its first large clinical trial. Neither Jonas Salk nor Albert Sabin received a Nobel Prize for their work in creating vaccines.
- I started my career as a malaria researcher, and I longed for the day that we would have an effective vaccine against this ancient and terrible disease. And today is that day, an historic day. Today, the WHO is recommending the broad use of the world’s first malaria vaccine.
- Tedros "WHO endorses use of world’s first malaria vaccine in Africa" (October 6,2021)
- Vaccination against destination-specific diseases plays an important role in preparing a traveler, although vaccine preventable diseases (VPDs) are rare in returning persons. While mandatory vaccinations in international travel are restricted to yellow fever, meningococcal meningitis (hajj), and rarely other vaccinations in special outbreak situations, recommendation for individual precaution by vaccination is based on data from returning travelers and from the epidemiologic situation in the visiting country. Weighing risk of vaccination against the benefit for the traveler is a prerequisite for pro/con decisions and implies de-tailed evaluation of personal risk of contracting a VPD. However, the currently licensed vaccines indicated for an international traveler are considered safe, well tolerated, and efficacious.
- Joseph Torresi, Herwig Kollaritsch, “Recommended/Required Travel Vaccines”, in Travel Medicine (Fourth Edition), (2019)
- They have to get the shots. The vaccinations are so important. This is really going around now. They have to get their shots.
- Donald Trump, reported in Maegan Vazquez, "Trump now says parents must vaccinate children in face of measles outbreak", CNN (April 26, 2019).
- Vaccine comes from the Latin word for “cow,” vacca, because the term was initially used to refer to inoculation using doses of w:cowpox that, it was discovered, protect humans against smallpox. This word is a relatively recent one in English, dating back to the 1880s
- The word vaccine was about much more than medicine in 2021. For many, the word symbolized a possible return to the lives we led before the pandemic. But it was also at the center of debates about personal choice, political affiliation, professional regulations, school safety, healthcare inequality, and so much more
- Back in the 2000s, we performed a series of studies in mice and people to understand how individual bouts of exercise and exercise training affect influenza infection and vaccination, respectively. In our animal studies, we found that moderate endurance exercise (30 min/day) could protect mice from death due to influenza. Mice that exercised for longer durations (∼2.5 h/day) exhibited an increase in some illness symptoms, but there was no statistically significant difference in mortality when compared to sedentary mice. We concluded that moderate exercise could be beneficial and that prolonged exercise could be detrimental to influenza-infected mice. For obvious reasons, we have not performed this experiment in people.
We also did a large study to determine whether 10 months of regular endurance exercise could improve influenza vaccination responses in older adults, a group that is at risk for infectious disease due to immunosenescence. We found that regular, moderate cardiovascular exercise could extend the protective effect of the annual influenza vaccination so that it maintained protective levels of antibodies throughout the entire influenza season (i.e., into March and April in the northern hemisphere). We concluded that regular moderate endurance exercise might be one way to boost the protective effect of annual influenza vaccination. It is very important for all people to receive the annual influenza vaccine.
- Jeffrey A. Woods, as quoted by Weimo Zhu in “Should, and how can, exercise be done during a coronavirus outbreak? An interview with Dr. Jeffrey A. Woods”, J Sport Health Sci. (2020 Mar Published online 2020 Feb 4); 9(2): 105–107.
- 'Flying vaccinator' is the concept of using genetically engineered hematophagous insects to deliver vaccines. Here we show the generation of a transgenic anopheline mosquito that expresses the Leishmania vaccine candidate, SP15, fused to monomeric red fluorescent protein (mDsRed) in its salivary glands. Importantly, mice bitten repeatedly by the transgenic mosquitoes raised anti‐SP15 antibodies, indicating delivery of SP15 via blood feeding with its immunogenicity intact. Thus, this technology makes possible the generation of transgenic mosquitoes that match the original concept of a ‘flying vaccinator’. However, medical safety issues and concerns about informed consent mitigate the use of the ‘flying vaccinator’ as a method to deliver vaccines.
- D. S. Yamamoto, H. Nagumo, S. Yoshida, “Flying vaccinator; a transgenic mosquito delivers a Leishmania vaccine via blood feeding”, Insect Molecular Biology, (04 May 2010).
"A Vaccine for the World”: U.S. Scientists Develop Low-Cost Shot to Inoculate Global South, Peter Hotez and Amy Goodman, Democracy Now!], January 03, 2022Edit
- Peter Hotez: The reason why we have this situation now with Omicron... is we allowed large unvaccinated populations in low- and middle-income countries to remain unvaccinated. Delta arose out of an unvaccinated population in India in early 2021, and Omicron out of a large unvaccinated population on the African continent later in the same year. So, these two variants of concern represent failures, failures by global leaders to work with sub-Saharan Africa, Southeast Asia and Latin America to vaccinate the Southern Hemisphere, vaccinate the Global South....
- Myself... Dr. Bottazzi... and our team of 20 scientists... make vaccines for diseases that the pharma companies won’t make... the only thing we know how to do is make low-cost, straightforward vaccines for use in resource-poor settings.... it was very difficult to get funding. We got no support from Operation Warp Speed, no support really from the G7 countries...
- We’ve licensed our prototype vaccine, and help in the co-development, to India, Indonesia, Bangladesh and now Botswana.... it’s really exciting to show that, you know, you don’t need to be a multinational pharmaceutical company and just make brand-new technologies that will only be suitable for the Northern Hemisphere. We can really make a vaccine for the world.
- We invite scientists from all over the world to come into our vaccine labs to learn how to make vaccines under a quality umbrella, whereas you cannot walk into Merck or GSK or Pfizer or Moderna and say, “Show me how to make a vaccine.” With our group, we can.... the biggest frustration was never really getting that support from the G7 countries... I going on cable news networks... trying to raise meager funds just to get started... fortunately, we were able to get some funding through Texas- and New York-based philanthropies, and...we raised about...$7 million...with that, we were able to pay our scientists to actually do this, transfer the technology, no patent, no strings attached, to India, now, as I said, Indonesia, Bangladesh and Botswana... we’ve been getting calls for help all over the world from ministries of science and ministries of health, and we do what we can. We could do a lot — I mean, if we had even a fraction of the support that, say, Moderna or the other pharma companies had gotten, who knows? We might have been able to have the whole world vaccinated by now....
- It’s even a vegan vaccine... So, now our partners in Indonesia... are trying to do this as a halal vaccine for Muslim-majority countries, which is pretty exciting, as well.
- Amy Goodman: Two hundred thousand Americans needlessly died because they believed disinformation from the far right, you tweeted. However, vaccine hesitancy seems to span the political divide, with left-leaning parents, some refusing to vaccinate themselves or their kids. Your message to those who think vaccines are a profit-making mechanism for Big Pharma that will pollute their bodies and irreversibly alter their immune system’s natural responses?
- Peter Hotez:...Vaccines clearly have been profitable for Pfizer and Moderna... but they can still save your life. ...And we’ve seen that of those 200,000 Americans who’ve died since June 1, we now know that 85% were unvaccinated, the other 15% split between partially vaccinated and a few full vaccinated, especially if they were immunocompromised or of extremely high age. But, overwhelmingly, it’s the unvaccinated who are losing their lives.
- And, overwhelmingly, that is coming from an aggressive campaign of disinformation, what I call anti-science aggression, coming from the conservative news outlets, coming from the members of Congress. You talked about Congresswoman Marjorie Taylor Greene just being taken off Twitter. That’s in part because she’s been out there at the CPAC conference and elsewhere discrediting vaccines, she and her colleagues. So we have about a half a dozen members of the U.S. Congress going out of their way to discredit the safety of vaccines, even saying they’re political instruments of control, or ridiculous things like, “First they’re going to vaccinate you, and then they’re going to take away your guns and your Bibles.” And as absurd as that sounds to us, there’s a fourth of the country that actually believes it, and those are the ones who are not getting vaccinated. And we even have far-right think tanks to give these far-right groups intellectual cover, academic cover. So, this is a whole ecosystem coming from political extremism on the far right, and it’s a killer. I’ve written an article called “Anti-science kills,” because now it’s killed 200,000 Americans since last June.
"As Omicron Spreads, 100+ Firms in Africa, Asia & Latin America Can Make mRNA Vaccine If Tech Shared", Democracy Now!, (16 December 2021) (full interview video, sound file, & transcript)
- As the coronavirus variant Omicron spreads across the world at an unprecedented rate, a group of vaccine experts has just released a list of over 100 companies in Africa, Asia and Latin America with the potential to produce mRNA vaccine. They say it is the one of the most viable solutions to fight vaccine inequity around the world and combat the spread of coronavirus variants, including Omicron. Democracy Now!
- The new coronavirus variant Omicron is spreading across the world at an unprecedented rate. The World Health Organization warns cases of the heavily mutated variant have been confirmed in 77 countries, and likely many others that have yet to detect it.
With international infections climbing, the Biden administration is facing renewed demands to follow through on his now seven-month-old pledge to ensure companies waive intellectual property protections on coronavirus vaccines and share them with the world.
Now a group of vaccine experts has just released a list of over a hundred companies in Africa, Asia and Latin America with the potential to produce mRNA vaccines to fight COVID-19. They say it’s one of the most viable solutions to fight vaccine inequity around the world and combat the spread of coronavirus variants, including Omicron. ~ (Amy Goodman)
- The backdrop to our report is Omicron. And what Omicron means, as we still figure out how infectious it is, how severe the infection that it causes is, what we know already are a few things. We know that all existing double-dose vaccines work less well against Omicron, which means that those who’ve had two doses of a Pfizer or Moderna vaccine in the United States need a booster. What we also know is that it’s highly transmissible and that it’s inevitably going to lead to a surge in cases, regardless of how severe they are.
What that in turn means for existing vaccine inequity, which is pretty deep — Nigeria has less than 2% of its population vaccinated as compared to countries in southern Europe, where the percentage is in the eighties — what it means is that vaccine inequity suddenly becomes worse. Why? Because everyone now needs more vaccines. ~ Achal Prabhala
- Our report is on mRNA vaccines, because they are a remarkable technology that we haven’t yet fully understood, meaning that they are not biology-based. They don’t require cells to be grown. And it means, therefore, that they can be made faster, more easily, and, therefore, by more companies than could make the previous vaccines we used to use before 2020.
We worked on finding companies that have the facilities and the quality standards and meet the technical requirements to make mRNA vaccines. And we found, to our astonishment, that there are at least 120 companies across Africa, Asia and Latin America who could be producing millions of doses of these vaccines, which, unfortunately, in the situation that we’re in — at a precipice — is really the only way by which we can get billions more vaccines into the world in the next three to six months. ~ Achal Prabhala
- These vaccines were created through public money — nearly $500 million of German public money from taxpayers to BioNTech, nearly a billion dollars in money from U.S. taxpayers through the government to Moderna, several billions of dollars after that in exchange for buying back vaccines at high prices. So these are very much the people’s vaccines. It’s just that they are private property.... when the Moderna CEO says, “Oh, anyone can make the Moderna vaccine,” he’s being a bit disingenuous... It’s not really possible to do that. The way vaccines work and the way regulation around vaccines work is that they need to be made with authorization and a license. Moderna and Pfizer or BioNTech... need to authorize companies to make their vaccine... to share an instruction manual as to how to do it... The problem is... it loosens Moderna and Pfizer and BioNTech’s stranglehold on these vaccines... It undercuts the massive tens of billions of dollars of profit and revenue that they can earn off selling to poor countries in the next couple of years, once they’re done with rich countries... which is why... ~ Achal Prabhala
- We’re asking the U.S. and German governments instead to say, “Look, in the face of this intransigence, it’s time to use emergency laws... that you can use, that you have the moral and legal power to put into effect, and end this pandemic for us and bring us out of this incredible cycle of hell. ~ Achal Prabhala
“How COVID unlocked the power of RNA vaccines” (12 January, 2021)Edit
Elie Doglin, “How COVID unlocked the power of RNA vaccines”, Nature, 589, (12 January 2021), pp.189-191.
- It was a Friday afternoon in March 2013 when Andy Geall got the call. Three people in China had just become infected with a new strain of avian influenza. The global head of vaccines research at Novartis, Rino Rappuoli, wanted to know whether Geall and his colleagues were ready to put their new vaccine technology to the test.
A year earlier, Geall’s team at Novartis’s US research hub in Cambridge, Massachusetts, had packaged strings of RNA nucleotides inside of small fat droplets, known as lipid nanoparticles (LNPs), and used them to successfully vaccinate rats against a respiratory virus1. Could they now do the same for the novel flu strain? And could they do it as fast as possible?
As Geall, head of the RNA group, recalls: “I said, ‘Yeah, sure. Just send us the sequence.’” By Monday, the team had begun synthesizing the RNA. By Wednesday, they were assembling the vaccine. By the weekend, they were testing it in cells — a week later, in mice.
The development happened at a breakneck speed. The Novartis team had achieved in one month what typically took a year or more.
But at the time, the ability to manufacture clinical-grade RNA was limited. Geall and his colleagues would never find out whether this vaccine, and several others that they developed, would work in people. In 2015, Novartis sold its vaccines business.
- The idea of using RNA in vaccines has been around for nearly three decades. More streamlined than conventional approaches, the genetic technology allows researchers to fast-track many stages of vaccine research and development. The intense interest now could lead to solutions for particularly recalcitrant diseases, such as tuberculosis, HIV and malaria. And the speed at which they can be made could improve seasonal-flu vaccines.
But future applications of the technology will run up against some challenges. The raw materials are expensive. Side effects can be troubling. And distribution currently requires a costly cold chain — the Pfizer–BioNTech COVID-19 vaccine, for example, must be stored at −70 °C. The urgency of COVID-19 is likely to speed up progress on some of those problems, but many companies might abandon the strategy once the current crisis subsides. The question remains: where will it end up?
- Vaccines teach the body to recognize and destroy disease-causing agents. Typically, weakened pathogens or fragments of the proteins or sugars on their surfaces, known as antigens, are injected to train the immune system to recognize an invader. But RNA vaccines carry only the directions for producing these invaders’ proteins. The aim is that they can slip into a person’s cells and get them to produce the antigens, essentially turning the body into its own inoculation factory.
The idea for RNA-based vaccination dates back to the 1990s, when researchers in France (at what is now the drug firm Sanofi Pasteur) first used RNA encoding an influenza antigen in mice. It produced a response, but the lipid delivery system that the team used proved too toxic to use in people. It would take another decade before companies looking at RNA-interference therapeutics — which rely on RNA’s ability to selectively block the production of specific proteins — discovered the LNP technologies that would make today’s COVID-19 vaccines possible.
- In 2012, around the time that Geall and his colleagues described1 the first LNP-encapsulated RNA vaccine, the US Defense Advanced Research Projects Agency (DARPA) began funding groups at Novartis, Pfizer, AstraZeneca, Sanofi Pasteur and elsewhere to work on RNA-encoded vaccines and therapeutics. None of the big-name firms stuck with the technology, however. “They were reticent about taking on any risk with a new regulatory pathway for vaccines, even though the data looked good,” says Dan Wattendorf, a former programme manager at DARPA.
- That was the full extent of clinical development for RNA vaccines at the beginning of 2020: only a dozen candidates had gone into people; four were swiftly abandoned after initial testing; and only one, for cytomegalovirus, had progressed to a larger, follow-on study.
Then came the coronavirus — and with it, “there’s been this enormous spotlight”, says Kristie Bloom, a gene-therapy researcher at the University of the Witwatersrand in Johannesburg, South Africa. In the past ten months alone, at least six RNA-based COVID-19 vaccines have entered human testing. Several more are nearing the clinic.
- [W]ith RNA vaccines making headlines, Geall and many of his former colleagues have been replaying their days at Novartis. Had the company not sold off its vaccines unit, could they have helped to stamp out Ebola or Zika outbreaks in the past decade?
“There’s always a little bit of sadness looking back,” says Christian Mandl, former head of research and early clinical development at Novartis’s vaccines unit. But he takes solace in the success of the COVID-19 vaccines today. “I am very proud that we made a valuable contribution.”
"The origins of vaccination" (28 Sept, 2020)Edit
Alexandra Flemming, “The origins of vaccination”, Nature, (28 Sept, 2020)
- Edward Jenner (1749–1823), a physician from Gloucestershire in England, is widely regarded as the ‘father of vaccination’ (Milestone 2). However, the origins of vaccination lie further back in time and also further afield. In fact, at the time Jenner reported his famous story about inoculating young James Phipps with cowpox and then demonstrating immunity to smallpox, the procedure of ‘variolation’ (referred to then as ‘inoculation’), by which pus is taken from a smallpox blister and introduced into a scratch in the skin of an uninfected person to confer protection, was already well established.
Variolation had been popularized in Europe by the writer and poet Lady Mary Wortley Montagu, best known for her ‘letters from the Ottoman Empire’. As wife of the British ambassador to Turkey, she had first witnessed variolation in Constantinople in 1717, which she mentioned in her famous ‘letter to a friend’. The following year, her son was variolated in Turkey, and her daughter received variolation in England in 1721. The procedure was initially met with much resistance — so much so that the first experimental variolation in England (including subsequent smallpox challenge) was carried out on condemned prisoners, who were promised freedom if they survived (they did). Nevertheless, the procedure was not without danger and subsequent prominent English variolators devised different techniques (often kept secret) to improve variolation, before it was replaced by the much safer cowpox ‘vaccination’ as described by Jenner.
- But how did variolation emerge in the Ottoman Empire? It turns out that at the time of Lady Montagu’s letter to her friend, variolation, or rather inoculation, was practised in a number of different places around the world. In 1714, Dr Emmanuel Timmonius, resident in Constantinople, had described the procedure of inoculation in a letter that was eventually published by the Philosophical Transactions of the Royal Society (London). He claimed that “the Circassians, Georgians, and other Asiatics” had introduced this practice “among the Turks and others at Constantinople”. His letter triggered a reply from Cotton Maher, a minister in Boston, USA, who reported that his servant Onesimus had undergone the procedure as a child in what is now southern Liberia, Africa. Moreover, two Welsh doctors, Perrot Williams and Richard Wright, reported that inoculation was well known in Wales and had been practised there since at least 1600.
Patrick Russell, an English doctor living in Aleppo (then part of the Ottoman Empire), described his investigations into the origins of inoculation in a letter written in 1786. He had sought the help of historians and doctors, who agreed that the practice was very old but was completely missing from written records. Nevertheless, it appears that at the time, inoculation was practised independently in several parts of Europe, Africa and Asia. The use of the needle (and often pinpricks in a circular pattern) was a common feature, but some places had other techniques: for example, in Scotland, smallpox-contaminated wool (a ‘pocky thread’) was wrapped around a child’s wrist, and in other places, smallpox scabs were placed into the hand of a child in order to confer protection. Despite the different techniques used, the procedure was referred to by the same name — ‘buying the pocks’ — which implies that inoculation may have had a single origin.
- Two places in particular have been suggested as the original ‘birthplace of inoculation’: India and China. In China, written accounts of the practice of ‘insufflation’ (blowing smallpox material into the nose) date to the mid-1500s. However, there are claims that inoculation was invented around 1000 ad by a Taoist or Buddhist monk or nun and practised as a mixture of medicine, magic and spells, covered by a taboo, so it was never written down.
Meanwhile, in India, 18th century accounts of the practice of inoculation (using a needle) trace it back to Bengal, where it had apparently been used for many hundreds of years. There are also claims that inoculation had in fact been practised in India for thousands of years and is described in ancient Sanscrit texts, although this has been contested.
”George Washington and the First Mass Military Inoculation” (February 12, 2009)Edit
Amy Lynn Filsinger & Raymond Dwek; ”George Washington and the First Mass Military Inoculation”, Library of Congress, (February 12, 2009)
- Traditionally, the Battle of Saratoga is credited with tipping the revolutionary scales. Yet the health of the Continental regulars involved in battle was a product of the ambitious initiative Washington began earlier that year at Morristown, close on the heels of the victorious Battle of Princeton. Among the Continental regulars in the American Revolution, 90 percent of deaths were caused by disease, and Variola the small pox virus was the most vicious of them all. (Gabriel and Metz 1992, 107)
On the 6th of January 1777, George Washington wrote to Dr. William Shippen Jr., ordering him to inoculate all of the forces that came through Philadelphia. He explained that: "Necessity not only authorizes but seems to require the measure, for should the disorder infect the Army . . . we should have more to dread from it, than from the Sword of the Enemy." The urgency was real. Troops were scarce and encampments had turned into nomadic hospitals of festering disease, deterring further recruitment. Both Benedict Arnold and Benjamin Franklin, after surveying the havoc wreaked by Variola in the Canadian campaign, expressed fears that the virus would be the army's ultimate downfall. (Fenn 2001, 69)
At the time, the practice of infecting the individual with a less-deadly form of the disease was widespread throughout Europe. Most British troops were immune to Variola, giving them an enormous advantage against the vulnerable colonists. (Fenn 2001, 131) Conversely, the history of inoculation in America (beginning with the efforts of the Reverend Cotton Mather in 1720) was pocked by the fear of the contamination potential of the process. Such fears led the Continental Congress to issue a proclamation in 1776 prohibiting Surgeons of the Army to inoculate.
Washington suspected the only available recourse was inoculation, yet contagion risks aside, he knew that a mass inoculation put the entire army in a precarious position should the British hear of his plans. Moreover, Historians estimate that less than a quarter of the Continental Army had ever had the virus; inoculating the remaining three quarters and every new recruit must have seemed daunting. Yet the high prevalence of disease among the army regulars was a significant deterrent to desperately needed recruits, and a dramatic reform was needed to allay their fears.
Weighing the risks, on February 5th of 1777, Washington finally committed to the unpopular policy of mass inoculation by writing to inform Congress of his plan. Throughout February, Washington, with no precedent for the operation he was about to undertake, covertly communicated to his commanding officers orders to oversee mass inoculations of their troops in the model of Morristown and Philadelphia (Dr. Shippen's Hospital). At least eleven hospitals had been constructed by the year's end.
Variola raged throughout the war, devastating the Native American population and slaves who had chosen to fight for the British in exchange for freedom. Yet the isolated infections that sprung up among Continental regulars during the southern campaign failed to incapacitate a single regiment. With few surgeons, fewer medical supplies, and no experience, Washington conducted the first mass inoculation of an army at the height of a war that immeasurably transformed the international system.
“Synthetic DNA vaccines: improved vaccine potency by electroporation and co-delivered genetic adjuvants” (Nov 4, 2013)Edit
Seleeke Flingai, Matias Czerwonko, Jonathan Goodman, Sagar B Kudchodkar, Kar Muthumani, David B Weiner; “Synthetic DNA vaccines: improved vaccine potency by electroporation and co-delivered genetic adjuvants”, Front Immunol'. 2013 Nov 4;4:354.
- In recent years, DNA vaccines have undergone a number of technological advancements that have incited renewed interest and heightened promise in the field. Two such improvements are the use of genetically engineered cytokine adjuvants and plasmid delivery via in vivo electroporation (EP), the latter of which has been shown to increase antigen delivery by nearly 1000-fold compared to naked DNA plasmid delivery alone. Both strategies, either separately or in combination, have been shown to augment cellular and humoral immune responses in not only mice, but also in large animal models. These promising results, coupled with recent clinical trials that have shown enhanced immune responses in humans, highlight the bright prospects for DNA vaccines to address many human diseases.
- Prevention is the most foolproof method of medical intervention, and the vaccine is its most representative example. Since Edward Jenner’s pioneering smallpox vaccine, vaccinology has followed an irregular path to its modern day form, with alternating periods of progress and stagnation (1, 2). Through advances in molecular biology, vaccinology has evolved from using basic inoculations of whole microorganisms to harnessing the power and flexibility of genetic engineering (3). DNA vaccination, one of the latest biotechnological breakthroughs, is the beginning of a new chapter in vaccine technology.
- The fundamental idea behind DNA vaccines (also known as genetic vaccines) is to induce immune responses against recombinant antigens encoded by genetically engineered DNA plasmids expressed in vivo. After immunization, host cellular machinery facilitates the expression of plasmid-encoded genes, which leads to the generation of foreign antigens that can be processed and presented on both major histocompatibility complex (MHC) class I and class II molecules. These host-synthesized foreign antigens can be recognized by the immune system, inducing a complete and effective immunization.
This novel method of vaccination was engineered in response to a series of emerging diseases that remain without proper prophylactic and therapeutic treatment. More than 50 years ago, pioneering studies carried out by Atanasiu et al. and Orth et al. showed that inoculation of mouse-derived tumor DNA induced tumors and led to seroconversion in injected mice (4, 5). The work of Wolff et al. showed that DNA plasmids injected intramuscularly (i.m.) could generate long-term gene expression in vivo without the need for a special delivery system (6); this finding helped generate much excitement for the scientific community. Within the past decade, four successful DNA plasmid products have been licensed for animal use: one for the treatment of West Nile virus in horses (7), one against hematopoietic necrosis virus in salmon (8), one for the treatment of melanoma in dogs (9), and a growth hormone-releasing hormone (GHRH) gene therapy for swine (10). However, despite promising studies in small animal models and improved efficacy in large animal models, the clinical ability of DNA vaccines still remains unproven. While the reasons for this inconsistency have yet to be fully elucidated, several attempts have been made to enhance immunogenicity in humans, resulting in studies that have provided a wealth of constructive information that may guide research efforts toward the development of improved DNA products.
- The seeds of DNA vaccinology were planted in the mid-twentieth century, when studies by Stasney et al. (11), Paschkis et al. (12), and Ito (13) demonstrated the ability to transfer DNA to animal cells by injection of crude DNA preparations isolated from tumors. These reports and others laid the groundwork for DNA vaccines by showing that DNA injection into animals can result in the expression of the delivered genes in vivo. However, perhaps the most important aspect of these early studies was that the immune system could respond to the gene products generated by DNA inoculation. For example, Atanasiu et al. (4) and Orth et al. (5) purified DNA extracts from polyoma viruses and demonstrated both tumor induction and the generation of anti-polyoma antibodies in injected animals. These findings were extended in studies by Israel and colleagues, who observed that injection of recombinant purified polyoma virus DNA resulted in tumor formation and anti-polyoma antibody production (14). Will and coworkers also observed humoral immune responses after inoculation of recombinant purified hepatitis B viral DNA into chimpanzees (15).
While many of these initial studies primarily focused on studying viral DNA biology (with humoral immunity against the inoculated gene product being of secondary importance), later studies sought to specifically study plasmid gene expression in vivo for a variety of applications. For example, Benvenisty and Reshef delivered genes encoding insulin and human growth hormone (HGH) into newborn rats, resulting in their expression in vivo (16). Later, studies by Jon Wolff and colleagues demonstrated long-term expression of DNA plasmids injected intramuscularly in mice (6). And in 1992, Tang et al. directly studied the immune response in mice elicited by DNA inoculation of foreign proteins. Using a gene gun to shoot gold particles coated with HGH-encoding DNA into mouse skin, the researchers found detectable levels of antibodies against the hormone, thus reproducing the earlier work of Israel, Atanasiu and Orth but in a more controlled manner (17). At the annual Cold Spring Harbor Vaccine meeting in September 1992, the laboratories of Margaret Liu (Merck), Harriet Robinson (University of Massachusetts), and David Weiner (University of Pennsylvania) independently reported that plasmid delivery into small animals could induce antibodies and cytotoxic T lymphocytes (CTLs) against influenza virus (18, 19) or HIV (20). Together, these studies were instrumental in laying the groundwork for the DNA vaccine field.
- The main advantage of DNA vaccines is their ability to stimulate both the humoral and cellular arms of the adaptive immune system. In regards to humoral immunity, the generation of antibodies by B lymphocytes against invading pathogens is one of the most effective defenses mounted by the immune system. Vaccines that utilize live-attenuated microorganisms, killed viral particles, or recombinant viral proteins elicit the production of specific antibodies that bind superficial microbial structures on the target pathogen. Unfortunately, immunological pressure or imprecise genome replication can cause certain pathogens to accumulate mutations that reduces the effectiveness of antibodies originally generated against the pathogen. Typically, antibody responses generated by traditional vaccines target only the specific antigens found in the inoculum, and are poorly able to control similar pathogens that carry either subtle or gross changes to the antigen. Due to the ability to genetically modify the antigen encoded by DNA vaccines, the vaccine can be designed to contain the most highly conserved regions of the superficial, antibody-generating structures on a pathogen, providing a means to generate broadly neutralizing antibodies against pathogens such as HIV and the influenza virus.
Regarding cellular immunity, CTLs eradicate infected or malignant cells upon recognition of foreign antigens in complex with MHC class I molecules on the target cell. Live-attenuated microorganisms can enter cells, and their viral proteins can be processed and directed to the MHC class I pathway for presentation upon the cell surface and the subsequent induction of CTL-mediated adaptive immunity. DNA vaccines also enter cells and produce antigen that can be processed and presented via MHC class I; however, DNA vaccines eschew the reversion risks associated with live-attenuated microorganisms.
Another major advantage of the DNA vaccine model is its versatility. In addition to the prevention of infectious diseases, DNA vaccines may also be used to treat malignancies and autoimmune or genetic disorders. When used for cancer therapy, plasmid DNA encoding a tumor-associated antigen (TAA) can be designed to induce CTL responses against cancerous cells expressing the antigen (33). Concerning autoimmune disorders, DNA plasmids may encode immunomodulatory proteins that could tailor the immune response to the type and intensity needed to ameliorate conditions as common as juvenile diabetes or food allergies.
- Vaccines as a whole have maintained a very strong safety profile. Nevertheless, live-attenuated and inactivated pathogens used in traditional vaccines carry the potential to return to virulence, which may cause pathogenic infections in vivo (34, 35), particularly in immunocompromised individuals. DNA vaccines, on the other hand, do not use microorganisms and therefore avoid the risk of reversion. Additionally, frequent vaccine-induced side effects such as headache, fever, and transient pain have shown reduced rates with DNA vaccines (36). Investigations into the possibility of DNA vaccine plasmids integrating into the host chromosome have not shown relevant levels of integration to occur (37, 38). Furthermore, preclinical and clinical studies have not detected detrimental anti-vector autoimmunity (i.e., disease-causing anti-nuclear or anti-DNA antibodies) after DNA vaccination, making it possible to administer multiple doses of DNA vaccines without triggering an immune reaction to the plasmid vector (39, 40); such an immunization protocol may be particularly useful for therapeutic cancer vaccination, which relies on repeated boosting of T-cell responses to be effective. This is in contrast with viral or bacterial vectors, which often induce anti-vector immunity that prevents boosting with the same vector (41). Lastly, while there has been evidence of anti-DNA antibodies generated as a result of epitope spreading (42), these antibodies were found to be transient and, most importantly, purely innocuous in animal models (43).
- Of the many advancements in DNA vaccines that have drastically improved immunogenicity, plasmid delivery via in vivo EP has proven to be one of the more impactful enhancements. EP involves the application of brief electric pulses to the vaccination site after injection of plasmid DNA. Administering EP results in the formation of transient pores in the plasma membrane of cells at the injection site (71, 72), which allows macromolecules such as nucleic acids to enter the cytoplasm (73). While the mechanisms for plasmid delivery by EP are still incompletely understood, the procedure improves plasmid delivery by a factor of 10–1,000 fold over naked DNA delivery alone (74). After the cessation of the electric pulses, pore closure traps the macromolecules within the cytoplasm. Not only does EP mediate enhanced plasmid uptake, but it also increases DNA distribution throughout the tissue and causes a local inflammatory reaction, both of which contribute to a stronger immune response (75). Importantly, the safety profile of EP after DNA vaccination is very similar to that of DNA delivered without EP, with no increased risk of toxicity or integration of the DNA plasmid into transfected cells. The most common adverse event described in clinical trials involving DNA vaccination with EP was increased pain at the application site.
While directly translating enhanced plasmid delivery to improved gene expression and immune responses is not without difficulty, comparison studies using reporter gene systems or immunogenicity readouts have established a strong correlation between EP delivery and augmented gene expression and immune responses (Figure (Figure1)1) (30, 76, 77). Furthermore, these improvements in DNA vaccine expression and potency can be achieved at significantly lower doses than with naked DNA delivery alone. A number of preclinical studies in small and large animal models have generated a substantial profile on the application of EP with DNA vaccination. For example, administration of the HIV DNA vaccine ADVAX was shown to increase antigen-specific CD4+ and CD8+ T-cell responses in mice when delivered by EP (78). Additional preclinical studies in pigs (79), cows (80, 81), rabbits (82), and others have had similar positive results in their respective DNA vaccine models with EP. A recent study compared protective antibody responses in chickens given a DNA vaccine containing the hemagglutinin (HA) gene of the avian influenza H5N1 virus delivered with or without EP (83): of the chickens that had the vaccine delivered by EP, 100% showed complete protection (low viral load and absence of clinical symptoms and mortality), while only 20% of the chickens who received the vaccine without EP developed antibodies.
- Because low immunogenicity has been the major deterrent toward using DNA vaccines in large animals and humans, several approaches have been investigated to increase the intensity and duration of vaccine-induced immune responses. One popular strategy has been to create vaccine cocktails, which includes the DNA vaccine along with plasmids encoding immunomodulatory proteins. Such adjuvant-encoding genes can be delivered either as separate plasmids or as additional genes encoded by the antigen-encoding plasmid. Upon vaccination, cells transfected with the adjuvant-encoding plasmid can express and secrete the molecular adjuvant into the surrounding region, affecting local APCs and cells in the draining lymph node. The end result is long-lasting, low level production of immunomodulatory cytokines that can tailor the immune response to the demands of each particular pathogen. For example, protection from certain viruses, other intracellular pathogens, or tumors may benefit from the use of cytokines that induce Th1-type immunity, such as IL-2, IL-12, IL-18, and IFNγ, which all generally promote cell-mediated immune responses (104). Conversely, cytokines such as IL-4 and IL-5 may be useful against extracellular pathogens while IL-10 and transforming growth factor-β (TGFβ) may prove effective in treating autoimmune disorders that arise from aberrant cell-mediated immunity (104). And while the role of Th17 cells during infection varies from pathogen to pathogen, evidence suggests that this cell subtype assists in the resistance to a number of bacterial and parasitic infections such as Leishmania (105, 106), Pseudomonas aeruginosa (107), Mycobacterium tuberculosis (108), and others. Cytokines such as TGFβ and either IL-6 or IL-21 are required for Th17 differentiation and may be useful for directing a Th17-type immune response during vaccination. By raising the concentration of certain immunomodulatory proteins during the initiation or boosting of an immune response, one can selectively activate or inhibit the division of the immune system that would lead to the greatest immunological benefit.
- The early promise of DNA vaccination had been tempered by lackluster immune responses in large animal models and humans. However, technological advances in the last decade have generated renewed interest in the improved, synthetically designed, and newly formulated DNA vaccine, especially when delivered by enhanced EP systems. Improved plasmid delivery via in vivo adaptive EP and the use of genetic adjuvants (in particular as plasmid-encoded IL-12) have proven to be powerful enhancers of DNA vaccines. Not only have these strategies improved immune responses in a variety of preclinical vaccination studies, but increasing evidence is suggesting that these approaches can also augment immune responses in humans. Given the various advantages of DNA vaccines – their ease of design, strong safety record, and stability, amongst others – the enhancements in immune responses in large animal models and humans is incredibly encouraging for the viability of DNA vaccines as a competitive vaccine platform. To carry these promising results further, additional research is needed on novel adjuvants, the timing of adjuvant administration, and the combination of genetic adjuvants and EP for optimal vaccination protocols. The prospects for treatment and prevention of human and animal disease by DNA vaccines are exciting, and the continual refinement of these technologies bode well for the present and future of this vaccine field.
“Stanford students study the stories behind medical breakthroughs” (June 25, 2020)Edit
S. Lochlann Jain as qtd. in “Stanford students study the stories behind medical breakthroughs”, by Melissa De Witte, (June 25, 2020)
- “This history of vaccines is an incredibly interesting history of social relationships, colonial relationships, global relationships, and relationships between animals and humans,” said Jain, a professor of anthropology in the School of Humanities and Sciences.
For example, when British physician Edward Jenner developed the smallpox vaccine in the late 18th century, he used his gardener’s 8-year-old child as one of his test subjects – an action that demonstrates both the loose ethical standards and social hierarchies of Georgian England. Jain also pointed out that Jenner’s idea for inoculation came from countryside folklore that dairymaids’ exposure to cowpox made them immune to smallpox.
“Although Jenner is known as the father of vaccinations, the idea didn’t come from nowhere. He built on social knowledge of the time,” said Jain.
- While Jenner discovered a preventive measure against smallpox, it took over 150 years for his method to be administered in a way that was safe to vaccine recipients. For example, early experiments exposed test subjects to the virus to make sure the vaccine worked, which sometimes led to infection and even death from the very illness one was being inoculated against. In other instances, early doses of the vaccine were sometimes contaminated with other harmful agents, such as the bacterium that causes syphilis.
“This history raises a host of questions about how we think about what is effective,” said Elliott M. Reichardt, a PhD student in anthropology, who took the class. “It challenges this history that we have of Edward Jenner and that the development of the smallpox vaccine was just perfect. No – it was quite dangerous for a lot of people.”
Reichardt said he was also surprised to learn about other dangers scientists encountered as they developed vaccines for other diseases, including the polio vaccine. For example, throughout the 1940s and 1950s, researchers in the U.S. relied on monkey cell lines in vaccine production. However, due to inadequate sterilization procedures, millions of Americans were inadvertently infected with an animal virus, simian virus 40.
“What these examples reveal is the iterative nature of engineering and medical research,” said Reichardt. “It also illustrates how this complex past is at times neglected in explaining the origins of contemporary vaccinology.”
- [D]uring development of the polio vaccine in the 1950s, scientists and government officials put aside Cold War politics of the era in a global effort to eradicate the disease. Students studied the case of communist Hungary, which even during the midst of civilian uprisings lifted the Iron Curtain to allow scientific collaborations with the West, enabling the import of both the polio vaccine and iron lungs, tank respirators used to support breathing in polio patients.
“If there is the right disease, avenues for collaboration can open up politically that otherwise might seem impossible,” said Reichardt.
- As the class learned about the global challenges of combating smallpox and other diseases like yellow fever, polio, HIV/AIDS, Ebola and Zika, they had to reckon with a pandemic of their own: the novel coronavirus.
With their own world upended by uncertainty of a new virus, stories from previous outbreaks felt all too familiar – particularly how people grappled with an illness they knew little about. For example, during the polio outbreak in the 1950s, all that people knew about the disease was that children were particularly vulnerable. Out of fear, some parents forbade their children from going outdoors.
“We could understand it from a very personal, urgent kind of way,” said Jain, who is currently studying how HIV/AIDS first emerged in New York and San Francisco when little was known about the disease – other than that it was fatal.
“Having to personally weigh our own risks amid uncertainty gave us a new insight into how people may have made decisions about sociality before they understood what they were dealing with, in the polio epidemic or in the HIV epidemic,” Jain said. “The pandemic has been particularly difficult for people in non-normative families, such as young people and queer people. This has provided insight into the early days of HIV.”
As Reichardt adjusted to sheltering-in-place, he said he was consoled by the knowledge that this was by no means the first occasion in history when people had to confine themselves to prevent contagion.
“Humans have been isolating themselves for extended periods of time for very long periods of time,” said Reichardt, observing that Italians in the 14th century quarantined themselves as a way to thwart the bubonic plague. “It was reassuring to know society is not going to collapse and we will be fine. Humans are resilient and we’re going to get through this.”
“Religious Objections to the Measles Vaccine? Get the Shots, Faith Leaders Say” (April 26, 2019)Edit
“Religious Objections to the Measles Vaccine? Get the Shots, Faith Leaders Say”, by Donald G. McNeil Jr., The New York Times, (April 26, 2019)
- Vaccines, the activists say, contain ingredients made from pigs, dogs, monkeys and aborted fetuses. Indeed, most of those assertions are based in fact. Ingredient lists published by the Centers for Disease Control and Prevention and the Institute for Vaccine Safety at Johns Hopkins show that vaccines may contain these elements (although any residual DNA is present only at the parts-per-million level).
Nonetheless, vaccination is endorsed by top Jewish and Islamic scholars, and by the Vatican. Religious authorities have meticulously studied how vaccines are made and what is in them, and still have ruled that they do not violate Jewish, Islamic or Catholic law.
Although no vaccine is without side effects, immunization is one of the greatest advances in medicine. The World Health Organization estimates that vaccines have saved more than 10 million lives in just the last decade.
- Vaccines are highly purified, but they still may contain isolated cells or traces of DNA from the human or animal cells they were grown in. Those “growth media” include cell lines originally derived — often decades ago — from monkey or dog kidneys, moth caterpillars, |calf blood, or the immature tissues of aborted human fetuses. (The widely circulated assertion that vaccines contain rat DNA is untrue.)
Some vaccines grown in eggs or using dairy products contain residual egg or casein proteins. And in some vaccines, the manufacturers add small doses of gelatin made from pig skin to prevent damage from heat or freeze-drying.
As New York’s current measles outbreak has spread, the more obscure ingredients of the measles vaccine — which is often delivered in a shot mixed with vaccines against mumps, rubella and chickenpox — have become an issue for some Orthodox Jews.
- [K]osher dietary laws are “just a total nonissue” with regard to vaccines, said Dr. Naor Bar-Zeev, a professor of international health and vaccine science at the Johns Hopkins Bloomberg School of Public Health. “All these complex laws apply to food ingested by mouth and are not in any way relevant to injected material.”
- Vaccines against viral diseases are made from viruses, which are just protein shells containing short stretches of DNA or RNA and can multiply only when grown in broths of live cells. Those cells are unusual in that they must be “immortal” — that is, able to replicate for decades without suffering “cell death,” the aging process.
(The best-known example is HeLa cells, which were isolated from a tumor in a woman named Henrietta Lacks, who died in 1951. More than 50 million tons of HeLa cells have since been grown for use in cancer research.)
The cells also must be free of cancer and viruses, which is one reason the ancestor cells come from fetuses that have never been exposed to pathogens — fetuses that were removed in sterile surgical environments, not from miscarriages.
- Catholics, the Vatican said, must choose alternative vaccines if they exist and press vaccine companies to make alternatives.
Nonetheless, because there are no alternatives, the use of existing vaccines — particularly against rubella, the Vatican ruled — was “morally justified” because of the higher need to protect children and pregnant women.
The Vatican reiterated that position as recently as two years ago in guidelines for Catholic health workers.
Mormons, Episcopalians, Lutherans and many other Christian denominations endorse vaccines, require them in their schools and distribute them at their missionary hospitals.
High authorities of all other major religions back vaccination, according to a study by Dorit R. Reiss, a medical law expert at the University of California Hastings Law School, and a paper in the journal Vaccine by John D. Grabenstein, an employee of Merck’s vaccine division.
Among Buddhists, the Dalai Lama has personally given polio vaccine to children to further the world polio-eradication drive. One of the first accounts of variolation — an ancient form of smallpox prevention — was from an 11th century Buddhist nun, who blew ground smallpox scabs into the noses of her patients.
“mRNA vaccines — a new era in vaccinology” (12 January 2018)Edit
Norbert Pardi, Michael J. Hogan, Frederick W. Porter & Drew Weissman; “mRNA vaccines — a new era in vaccinology”, Nature Reviews Drug Discovery, (12 January 2018), volume 17, pp. 261–279
- mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use.
- Nucleic acid therapeutics have emerged as promising alternatives to conventional vaccine approaches. The first report of the successful use of in vitro transcribed (IVT) mRNA in animals was published in 1990, when reporter gene mRNAs were injected into mice and protein production was detected5. A subsequent study in 1992 demonstrated that administration of vasopressin-encoding mRNA in the hypothalamus could elicit a physiological response in rats6. However, these early promising results did not lead to substantial investment in developing mRNA therapeutics, largely owing to concerns associated with mRNA instability, high innate immunogenicity and inefficient in vivo delivery. Instead, the field pursued DNA-based and protein-based therapeutic approaches7,8.
Over the past decade, major technological innovation and research investment have enabled mRNA to become a promising therapeutic tool in the fields of vaccine development and protein replacement therapy. The use of mRNA has several beneficial features over subunit, killed and live attenuated virus, as well as DNA-based vaccines. First, safety: as mRNA is a non-infectious, non-integrating platform, there is no potential risk of infection or insertional mutagenesis. Additionally, mRNA is degraded by normal cellular processes, and its in vivo half-life can be regulated through the use of various modifications and delivery methods9,10,11,12. The inherent immunogenicity of the mRNA can be down-modulated to further increase the safety profile9,12,13. Second, efficacy: various modifications make mRNA more stable and highly translatable9,12,13. Efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm (reviewed in Refs 10,11). mRNA is the minimal genetic vector; therefore, anti-vector immunity is avoided, and mRNA vaccines can be administered repeatedly. Third, production: mRNA vaccines have the potential for rapid, inexpensive and scalable manufacturing, mainly owing to the high yields of in vitro transcription reactions.
- mRNA is the intermediate step between the translation of protein-encoding DNA and the production of proteins by ribosomes in the cytoplasm. Two major types of RNA are currently studied as vaccines: non-replicating mRNA and virally derived, self-amplifying RNA. Conventional mRNA-based vaccines encode the antigen of interest and contain 5′ and 3′ untranslated regions (UTRs), whereas self-amplifying RNAs encode not only the antigen but also the viral replication machinery that enables intracellular RNA amplification and abundant protein expression.
The construction of optimally translated IVT mRNA suitable for therapeutic use has been reviewed previously14,15. Briefly, IVT mRNA is produced from a linear DNA template using a T7, a T3 or an Sp6 phage RNA polymerase16. The resulting product should optimally contain an open reading frame that encodes the protein of interest, flanking UTRs, a 5′ cap and a poly(A) tail. The mRNA is thus engineered to resemble fully processed mature mRNA molecules as they occur naturally in the cytoplasm of eukaryotic cells.
Complexing of mRNA for in vivo delivery has also been recently detailed10,11. Naked mRNA is quickly degraded by extracellular RNases17 and is not internalized efficiently. Thus, a great variety of in vitro and in vivo transfection reagents have been developed that facilitate cellular uptake of mRNA and protect it from degradation. Once the mRNA transits to the cytosol, the cellular translation machinery produces protein that undergoes post-translational modifications, resulting in a properly folded, fully functional protein. This feature of mRNA pharmacology is particularly advantageous for vaccines and protein replacement therapies that require cytosolic or transmembrane proteins to be delivered to the correct cellular compartments for proper presentation or function. IVT mRNA is finally degraded by normal physiological processes, thus reducing the risk of metabolite toxicity.
- Various mRNA vaccine platforms have been developed in recent years and validated in studies of immunogenicity and efficacy18,19,20. Engineering of the RNA sequence has rendered synthetic mRNA more translatable than ever before. Highly efficient and non-toxic RNA carriers have been developed that in some cases21,22 allow prolonged antigen expression in vivo (Table 1). Some vaccine formulations contain novel adjuvants, while others elicit potent responses in the absence of known adjuvants. The following section summarizes the key advances in these areas of mRNA engineering and their impact on vaccine efficacy.
- Physical delivery methods in vivo. To increase the efficiency of mRNA uptake in vivo, physical methods have occasionally been used to penetrate the cell membrane. An early report showed that mRNA complexed with gold particles could be expressed in tissues using a gene gun, a microprojectile method69. The gene gun was shown to be an efficient RNA delivery and vaccination method in mouse models70,71,72,73, but no efficacy data in large animals or humans are available. In vivo electroporation has also been used to increase uptake of therapeutic RNA74,75,76; however, in one study, electroporation increased the immunogenicity of only a self-amplifying RNA and not a non-replicating mRNA-based vaccine74. Physical methods can be limited by increased cell death and restricted access to target cells or tissues. Recently, the field has instead favoured the use of lipid or polymer-based nanoparticles as potent and versatile delivery vehicles.
- Cationic lipid and polymer-based delivery. Highly efficient mRNA transfection reagents based on cationic lipids or polymers, such as TransIT-mRNA (Mirus Bio LLC) or Lipofectamine (Invitrogen), are commercially available and work well in many primary cells and cancer cell lines9,13, but they often show limited in vivo efficacy or a high level of toxicity (N.P. and D.W., unpublished observations). Great progress has been made in developing similarly designed complexing reagents for safe and effective in vivo use, and these are discussed in detail in several recent reviews10,11,79,80. Cationic lipids and polymers, including dendrimers, have become widely used tools for mRNA administration in the past few years. The mRNA field has clearly benefited from the substantial investment in in vivo small interfering RNA (siRNA) administration, where these delivery vehicles have been used for over a decade. Lipid nanoparticles (LNPs) have become one of the most appealing and commonly used mRNA delivery tools. LNPs often consist of four components: an ionizable cationic lipid, which promotes self-assembly into virus-sized (~100 nm) particles and allows endosomal release of mRNA to the cytoplasm; lipid-linked polyethylene glycol (PEG), which increases the half-life of formulations; cholesterol, a stabilizing agent; and naturally occurring phospholipids, which support lipid bilayer structure. Numerous studies have demonstrated efficient in vivo siRNA delivery by LNPs (reviewed in Ref. 81), but it has only recently been shown that LNPs are potent tools for in vivo delivery of self-amplifying RNA19 and conventional, non-replicating mRNA21. Systemically delivered mRNA–LNP complexes mainly target the liver owing to binding of apolipoprotein E and subsequent receptor-mediated uptake by hepatocytes82, and intradermal, intramuscular and subcutaneous administration have been shown to produce prolonged protein expression at the site of the injection21,22. The mechanisms of mRNA escape into the cytoplasm are incompletely understood, not only for artificial liposomes but also for naturally occurring exosomes83. Further research into this area will likely be of great benefit to the field of therapeutic RNA delivery.
- Development of prophylactic or therapeutic vaccines against infectious pathogens is the most efficient means to contain and prevent epidemics. However, conventional vaccine approaches have largely failed to produce effective vaccines against challenging viruses that cause chronic or repeated infections, such as HIV-1, herpes simplex virus and respiratory syncytial virus (RSV). Additionally, the slow pace of commercial vaccine development and approval is inadequate to respond to the rapid emergence of acute viral diseases, as illustrated by the 2014–2016 outbreaks of the Ebola and Zika viruses. Therefore, the development of more potent and versatile vaccine platforms is crucial.
Preclinical studies have created hope that mRNA vaccines will fulfill many aspects of an ideal clinical vaccine: they have shown a favourable safety profile in animals, are versatile and rapid to design for emerging infectious diseases, and are amenable to scalable good manufacturing practice (GMP) production (already under way by several companies). Unlike protein immunization, several formats of mRNA vaccines induce strong CD8+ T cell responses, likely owing to the efficient presentation of endogenously produced antigens on MHC class I molecules, in addition to potent CD4+ T cell responses56,87,88. Additionally, unlike DNA immunization, mRNA vaccines have shown the ability to generate potent neutralizing antibody responses in animals with only one or two low-dose immunizations20,22,85. As a result, mRNA vaccines have elicited protective immunity against a variety of infectious agents in animal models19,20,22,56,89,90 and have therefore generated substantial optimism. However, recently published results from two clinical trials of mRNA vaccines for infectious diseases were somewhat modest, leading to more cautious expectations about the translation of preclinical success to the clinic22,91 (discussed further below).
Two major types of RNA vaccine have been utilized against infectious pathogens: self-amplifying or replicon RNA vaccines and non-replicating mRNA vaccines. Non-replicating mRNA vaccines can be further distinguished by their delivery method: ex vivo loading of DCs or direct in vivo injection into a variety of anatomical sites. As discussed below, a rapidly increasing number of preclinical studies in these areas have been published recently, and several have entered human clinical trials (Table 2).
- Nucleoside-modified mRNA vaccines represent a new and highly efficacious category of mRNA vaccines. Owing to the novelty of this immunization platform, our knowledge of efficacy is limited to the results of four recent publications that demonstrated the potency of such vaccines in small and large animals. The first published report demonstrated that a single intradermal injection of LNP-formulated mRNA encoding Zika virus prM-E, modified with 1-methylpseudouridine and FPLC purification, elicited protective immune responses in mice and rhesus macaques with the use of as little as 50 μg (0.02 mg kg−1) of vaccine in macaques20. A subsequent study by a different group tested a similarly designed vaccine against Zika virus in mice and found that a single intramuscular immunization elicited moderate immune responses, and a booster vaccination resulted in potent and protective immune responses85. This vaccine also incorporated the modified nucleoside 1-methylpseudouridine, but FPLC purification or other methods of removing dsRNA contaminants were not reported. Notably, this report showed that antibody-dependent enhancement of secondary infection with a heterologous flavivirus, a major concern for dengue and Zika virus vaccines, could be diminished by removing a cross-reactive epitope in the E protein. A recent follow-up study evaluated the same vaccine in a model of maternal vaccination and fetal infection112. Two immunizations reduced Zika virus infection in fetal mice by several orders of magnitude and completely rescued a defect in fetal viability.
- mRNA-based cancer vaccines have been recently and extensively reviewed115,116,117,118,119. Below, the most recent advances and directions are highlighted. Cancer vaccines and other immunotherapies represent promising alternative strategies to treat malignancies. Cancer vaccines can be designed to target tumour-associated antigens that are preferentially expressed in cancerous cells, for example, growth-associated factors, or antigens that are unique to malignant cells owing to somatic mutation120. These neoantigens, or the neoepitopes within them, have been deployed as mRNA vaccine targets in humans121 (Box 2). Most cancer vaccines are therapeutic, rather than prophylactic, and seek to stimulate cell-mediated responses, such as those from CTLs, that are capable of clearing or reducing tumour burden122. The first proof-of-concept studies that not only proposed the idea of RNA cancer vaccines but also provided evidence of the feasibility of this approach were published more than two decades ago123,124. Since then, numerous preclinical and clinical studies have demonstrated the viability of mRNA vaccines to combat cancer (Table 3).
- As DCs are central players in initiating antigen-specific immune responses, it seemed logical to utilize them for cancer immunotherapy. The first demonstration that DCs electroporated with mRNA could elicit potent immune responses against tumour antigens was reported by Boczkowski and colleagues in 1996 (Ref. 124). In this study, DCs pulsed with ovalbumin (OVA)-encoding mRNA or tumour-derived RNAs elicited a tumour-reducing immune response in OVA-expressing and other melanoma models in mice. A variety of immune regulatory proteins have been identified in the form of mRNA-encoded adjuvants that can increase the potency of DC cancer vaccines. Several studies demonstrated that electroporation of DCs with mRNAs encoding co-stimulatory molecules such as CD83, tumour necrosis factor receptor superfamily member 4 (TNFRSF4; also known as OX40) and 4-1BB ligand (4-1BBL) resulted in a substantial increase in the immune stimulatory activity of DCs125,126,127,128. DC functions can also be modulated through the use of mRNA-encoded pro-inflammatory cytokines, such as IL-12, or trafficking-associated molecules129,130,131. As introduced above, TriMix is a cocktail of mRNA-encoded adjuvants (CD70, CD40L and constitutively active TLR4) that can be electroporated in combination with antigen-encoding mRNA or mRNAs132. This formulation proved efficacious in multiple preclinical studies by increasing DC activation and shifting the CD4+ T cell phenotype from T regulatory cells to T helper 1 (TH1)-like cells132,133,134,135,136. Notably, the immunization of patients with stage III or stage IV melanoma using DCs loaded with mRNA encoding melanoma-associated antigens and TriMix adjuvant resulted in tumour regression in 27% of treated individuals137. Multiple clinical trials have now been conducted using DC vaccines targeting various cancer types, such as metastatic prostate cancer, metastatic lung cancer, renal cell carcinoma, brain cancers, melanoma, acute myeloid leukaemia, pancreatic cancer and others138,139 (reviewed in Refs 51,58).
A new line of research combines mRNA electroporation of DCs with traditional chemotherapy agents or immune checkpoint inhibitors. In one trial, patients with stage III or IV melanoma were treated with ipilimumab, a monoclonal antibody against CTL antigen 4 (CTLA4), and DCs loaded with mRNA encoding melanoma-associated antigens plus TriMix. This intervention resulted in durable tumour reduction in a proportion of individuals with recurrent or refractory melanoma140.
- The route of administration and delivery format of mRNA vaccines can greatly influence outcomes. A variety of mRNA cancer vaccine formats have been developed using common delivery routes (intradermal, intramuscular, subcutaneous or intranasal) and some unconventional routes of vaccination (intranodal, intravenous, intrasplenic or intratumoural).
Intranodal administration of naked mRNA is an unconventional but efficient means of vaccine delivery. Direct mRNA injection into secondary lymphoid tissue offers the advantage of targeted antigen delivery to antigen-presenting cells at the site of T cell activation, obviating the need for DC migration. Several studies have demonstrated that intranodally injected naked mRNA can be selectively taken up by DCs and can elicit potent prophylactic or therapeutic antitumour T cell responses62,66; an early study also demonstrated similar findings with intrasplenic delivery141. Coadministration of the DC-activating protein FMS-related tyrosine kinase 3 ligand (FLT3L) was shown in some cases to further improve immune responses to intranodal mRNA vaccination142,143. Incorporation of the TriMix adjuvant into intranodal injections of mice with mRNAs encoding tumour-associated antigens resulted in potent antigen-specific CTL responses and tumour control in multiple tumour models133. A more recent study demonstrated that intranodal injection of mRNA encoding the E7 protein of human papillomavirus (HPV) 16 with TriMix increased the number of tumour-infiltrating CD8+ T cells and inhibited the growth of an E7-expressing tumour model in mice67.
The success of preclinical studies has led to the initiation of clinical trials using intranodally injected naked mRNA encoding tumour-associated antigens into patients with advanced melanoma (NCT01684241) and patients with hepatocellular carcinoma (EudraCT: 2012-005572-34). In one published trial, patients with metastatic melanoma were treated with intranodally administered DCs electroporated with mRNA encoding the melanoma-associated antigens tyrosinase or gp100 and TriMix, which induced limited antitumour responses144.
Intranasal vaccine administration is a needle-free, noninvasive manner of delivery that enables rapid antigen uptake by DCs. Intranasally delivered mRNA complexed with Stemfect (Stemgent) LNPs resulted in delayed tumour onset and increased survival in prophylactic and therapeutic mouse tumour models using the OVA-expressing E.G7-OVA T lymphoblastic cell line145.
- Intratumoural mRNA vaccination is a useful approach that offers the advantage of rapid and specific activation of tumour-resident T cells. Often, these vaccines do not introduce mRNAs encoding tumour-associated antigens but simply aim to activate tumour-specific immunity in situ using immune stimulatory molecules. An early study demonstrated that naked mRNA or protamine-stabilized mRNA encoding a non-tumour related gene (GLB1) impaired tumour growth and provided protection in a glioblastoma mouse model, taking advantage of the intrinsic immunogenic properties of mRNA146. A more recent study showed that intratumoural delivery of mRNA encoding an engineered cytokine based on interferon-β (IFNβ) fused to a transforming growth factor-β (TGFβ) antagonist increased the cytolytic capacity of CD8+ T cells and modestly delayed tumour growth in OVA-expressing lymphoma or lung carcinoma mouse models147. It has also been shown that intratumoural administration of TriMix mRNA that does not encode tumour-associated antigens results in activation of CD8α+ DCs and tumour-specific T cells, leading to delayed tumour growth in various mouse models148.
- Systemic administration of mRNA vaccines is not common owing to concerns about aggregation with serum proteins and rapid extracellular mRNA degradation; thus, formulating mRNAs into carrier molecules is essential. As discussed above, numerous delivery formulations have been developed to facilitate mRNA uptake, increase protein translation and protect mRNA from RNases10,11,79,80. Another important issue is the biodistribution of mRNA vaccines after systemic delivery. Certain cationic LNP-based complexing agents delivered intravenously traffic mainly to the liver21, which may not be ideal for DC activation. An effective strategy for DC targeting of mRNA vaccines after systemic delivery has recently been described59. An mRNA–lipoplex (mRNA–liposome complex) delivery platform was generated using cationic lipids and neutral helper lipids formulated with mRNA, and it was discovered that the lipid-to-mRNA ratio, and thus the net charge of the particles, has a profound impact on the biodistribution of the vaccine. While a positively charged lipid particle primarily targeted the lung, a negatively charged particle targeted DCs in secondary lymphoid tissues and bone marrow. The negatively charged particle induced potent immune responses against tumour-specific antigens that were associated with impressive tumour reduction in various mouse models59. As no toxic effects were observed in mice or non-human primates, clinical trials using this approach to treat patients with advanced melanoma or triple-negative breast cancer have been initiated (NCT02410733 and NCT02316457).
- A variety of antigen-presenting cells reside in the skin149, making it an ideal site for immunogen delivery during vaccination (Fig. 3). Thus, the intradermal route of delivery has been widely used for mRNA cancer vaccines. An early seminal study demonstrated that intradermal administration of total tumour RNA delayed tumour growth in a fibrosarcoma mouse model65. Intradermal injection of mRNA encoding tumour antigens in the protamine-based RNActive platform proved efficacious in various mouse models of cancer36 and in multiple prophylactic and therapeutic clinical settings (Table 3). One such study demonstrated that mRNAs encoding survivin and various melanoma tumour antigens resulted in increased numbers of antigen-specific T cells in a subset of patients with melanoma150. In humans with castration-resistant prostate cancer, an RNActive vaccine expressing multiple prostate cancer-associated proteins elicited antigen-specific T cell responses in the majority of recipients151. Lipid-based carriers have also contributed to the efficacy of intradermally delivered mRNA cancer vaccines. The delivery of OVA-encoding mRNA in DOTAP and/or DOPE liposomes resulted in antigen-specific CTL activity and inhibited growth of OVA-expressing tumours in mice152. In the same study, coadministration of mRNA encoding granulocyte–macrophage colony-stimulating factor (GM-CSF) improved OVA-specific cytolytic responses. Another report showed that subcutaneous delivery of LNP-formulated mRNA encoding two melanoma-associated antigens delayed tumour growth in mice, and co-delivery of lipopolysaccharide (LPS) in LNPs increased both CTL and antitumour activity153. In general, mRNA cancer vaccines have proved immunogenic in humans, but further refinement of vaccination methods, as informed by basic immunological research, will likely be necessary to achieve greater clinical benefits.
- Once the mRNA is synthesized, it is processed though several purification steps to remove reaction components, including enzymes, free nucleotides, residual DNA and truncated RNA fragments. While LiCl precipitation is routinely used for laboratory-scale preparation, purification at the clinical scale utilizes derivatized microbeads in batch or column formats, which are easier to utilize at large scale156,157. For some mRNA platforms, removal of dsRNA and other contaminants is critical for the potency of the final product, as it is a potent inducer of interferon-dependent translation inhibition. This has been accomplished by reverse-phase FPLC at the laboratory scale158, and scalable aqueous purification approaches are being investigated. After mRNA is purified, it is exchanged into a final storage buffer and sterile-filtered for subsequent filling into vials for clinical use. RNA is susceptible to degradation by both enzymatic and chemical pathways157. Formulation buffers are tested to ensure that they are free of contaminating RNases and may contain buffer components, such as antioxidants and chelators, which minimize the effects of reactive oxygen species and divalent metal ions that lead to mRNA instability159.
- Several different mRNA vaccines have now been tested from phase I to IIb clinical studies and have been shown to be safe and reasonably well tolerated (Tables 2, 3). However, recent human trials have demonstrated moderate and in rare cases severe injection site or systemic reactions for different mRNA platforms22,91. Potential safety concerns that are likely to be evaluated in future preclinical and clinical studies include local and systemic inflammation, the biodistribution and persistence of expressed immunogen, stimulation of auto-reactive antibodies and potential toxic effects of any non-native nucleotides and delivery system components. A possible concern could be that some mRNA-based vaccine platforms54,166 induce potent type I interferon responses, which have been associated not only with inflammation but also potentially with autoimmunity167,168. Thus, identification of individuals at an increased risk of autoimmune reactions before mRNA vaccination may allow reasonable precautions to be taken. Another potential safety issue could derive from the presence of extracellular RNA during mRNA vaccination. Extracellular naked RNA has been shown to increase the permeability of tightly packed endothelial cells and may thus contribute to oedema169. Another study showed that extracellular RNA promoted blood coagulation and pathological thrombus formation170. Safety will therefore need continued evaluation as different mRNA modalities and delivery systems are utilized for the first time in humans and are tested in larger patient populations.
- The fast pace of progress in mRNA vaccines would not have been possible without major recent advances in the areas of innate immune sensing of RNA and in vivo delivery methods. Extensive basic research into RNA and lipid and polymer biochemistry has made it possible to translate mRNA vaccines into clinical trials and has led to an astonishing level of investment in mRNA vaccine companies (Table 4). Moderna Therapeutics, founded in 2010, has raised almost US$2 billion in capital with a plan to commercialize mRNA-based vaccines and therapies172,173. The US Biomedical Advanced Research and Development Authority (BARDA) has committed support for Moderna's clinical evaluation of a promising nucleoside-modified mRNA vaccine for Zika virus (NCT03014089). In Germany, CureVac AG has an expanding portfolio of therapeutic targets174, including both cancer and infectious diseases, and BioNTech is developing an innovative approach to personalized cancer medicine using mRNA vaccines121 (Box 2). The translation of basic research into clinical testing is also made more expedient by the commercialization of custom GMP products by companies such as New England Biolabs and Aldevron175. Finally, the recent launch of the Coalition for Epidemic Preparedness Innovations (CEPI) provides great optimism for future responses to emerging viral epidemics. This multinational public and private partnership aims to raise $1 billion to develop platform-based vaccines, such as mRNA, to rapidly contain emerging outbreaks before they spread out of control.
”History of vaccination” (Aug 26, 2014)Edit
Stanley Plotkin, ”History of vaccination”, Proc Natl Acad Sci U S A. 2014 Aug 26; 111(34): 12283–12287.
- Vaccines have a history that started late in the 18th century. From the late 19th century, vaccines could be developed in the laboratory. However, in the 20th century, it became possible to develop vaccines based on immunologic markers. In the 21st century, molecular biology permits vaccine development that was not possible before.
One of the brightest chapters in the history of science is the impact of vaccines on human longevity and health. Over 300 years have elapsed since the first vaccine was discovered.
- In current articles that describe novel technologies, it is often said that they will enable “rational” development of vaccines. The opposite of rational is irrational, but presumably the writers mean to contrast rational with “empiric.” However, in fact, vaccine development has been based on rational choices ever since the mid-20th century, when immunology advanced to the point of distinguishing protection mediated by antibody and that mediated by lymphocytes, and when passage in cell culture permitted the selection of attenuated mutants. After that point, successful vaccines have been “rationally” developed by protection studies in animals; by inference from immune responses shown to protect against repeated natural infection (the so-called mechanistic correlates of protection) (6); and from the use of passive administration of antibodies against specific antigens to show that those antigens should be included in vaccines.
- The idea of attenuation of virulent infections developed slowly over the course of centuries. Variolation was analogous to the use of small amounts of poison to render one immune to toxic effects. Jenner's use of an animal poxvirus (probably horsepox) to prevent smallpox was essentially based on the idea that an agent virulent for animals might be attenuated in humans (7). This idea played a role in the development of bacillus Calmette–Guérin but is even more obvious in the selection of rhesus and bovine rotavirus strains to aid the creation of human rotavirus vaccines as mentioned below under Reassortment.
It was Pasteur and his colleagues who most clearly formulated the idea of attenuation and demonstrated its utility, first with Pasteurella multocida, the cause of a diarrheal disease in chickens (8), then anthrax in sheep and most sensationally rabies virus in animals and humans (9). Their first approaches involved exposure to oxygen or heat, both of which played a role in the development of the rabies vaccine and in the famous anthrax challenge experiment at Pouilly-le-Fort (10). However, the more powerful technique of serial cultivation of a pathogen in vitro or in inhabitual hosts originated with Calmette and Guérin, who passaged bovine tuberculosis bacteria 230 times in artificial media to obtain an attenuated strain to protect against human tuberculosis (11). Later in the 20th century, Sellards and Laigret (12) and, more successfully, Theiler and Smith (13) attenuated yellow fever virus by serial passage in mice and in chicken embryo tissues, respectively.
- By the 1940s, virologists understood that attenuation could be achieved by passage in abnormal hosts. Notably, Hilary Koprowski and coworkers developed rabies and oral polio vaccines by passage in chicken embryo or mice (14, 15). However, this method was inefficient, and mice were not a sterile medium. A revolution happened with the discovery that cells could be cultured in vitro and used as substrates for viral growth. Enders, Weller, and Robbins (16) showed that many viruses could be grown in cell culture, including polio and measles, and this method was vigorously taken up by vaccine developers. The oral polio vaccine of Albert Sabin and the measles, rubella, mumps, and varicella vaccines were all made possible through selection of clones by cell-culture passage in vitro (17–21). In essence, passage in cell culture leads to adaptation to growth in that medium, and the mutants best capable of growth have often lost or modified the genes that allow them to infect and spread within a human host. The oral polio vaccine is a good example, in that the mutants that occur in cell-culture passage that confer inability to cause paralysis were isolated by selection of clones with low neurovirulence in monkeys. These mutations are at least partly lost after replication of attenuated strains in the human intestine, leading to rare cases of paralysis after vaccination (22). Adaptation of viruses to growth at temperatures below 37 °C, the normal temperature of humans, also is attenuating, as was the case for rubella vaccine (20). Another live vaccine, thus far used only in the military to prevent epidemic pneumonia, consists of adeno 4 and 7 viruses grown in human diploid cell strains and administered orally to replicate in the intestine (23). Other live vaccines attenuated in cell-culture passage are the monovalent rotavirus vaccine attenuated by passage in Vero cells (24) and the Japanese encephalitis strain SA14-14-2 (25).
- Certain RNA viruses have segmented genomes that can be manipulated in a way similar to the chromosomes of eukaryotes. Cocultivation of two viruses in cell culture with clone selection by plaque formation allows isolation of viruses with RNA segments from both viruses. Reassortment has enabled the creation of three major vaccines: live and inactivated influenza (26, 27), as well as one of the two rotavirus vaccines (28). In the case of inactivated influenza, the objective is to select the segments coding for hemagglutinin and neuraminidase and to combine them with segments coding for the internal genes of viruses that grow well. Thus, one obtains a vaccine virus that is safe to handle but still generates functional antibodies against virulent influenza strains.
- Another discovery toward the end of the 19th century was that immunogenicity could be retained if bacteria were carefully killed by heat or chemical treatment. The first inactivated vaccines were developed more or less simultaneously by Salmon and Smith in the United States and the Pasteur Institute group (Roux and Chamberland ) in France (32, 33). Inactivation was first applied to pathogens such as the typhoid, plague, and cholera bacilli. This era was marked by competition between French, German, and English workers to develop antibacterial vaccines. Inactivated vaccines against typhoid were first applied by Wright and Semple in England and Pfeiffer and Kolle in Germany (34, 35). Humans were vaccinated against plague by Haffkine, using inactivated plague bacilli (36). Live vaccines against cholera were developed by Ferran in Spain and Haffkine in France (37), but it was ultimately the vaccine developed by Kolle using heat-inactivated cholera bacilli that came into general use (38). That vaccine was given parenterally but was painful and did not give long-lasting immunity. More recently, a vaccine was developed that consists of orally administered killed cholera bacteria, with or without the B subunit of cholera toxin (39). Formalin-inactivated whole-cell pertussis vaccine was first tested by Madsen (40) and was later shown to be relatively successful in controlling serious disease (41). However, it was the later work of Kendrick and Eldering that permitted standardization and safety of a whole-cell vaccine (42).
In 1923, Glenny and Hopkins made diphtheria toxin less toxic by formalin treatment (43). Ramon improved on this discovery and showed it was possible to inactivate the toxicity of those molecules yet retain their ability to induce toxin-neutralizing antibodies (44).
In the 20th century, chemical inactivation was also applied to viruses. Influenza vaccine was the first successful inactivated virus vaccine (45), and experience with that vaccine served Salk well in his successful effort to develop an inactivated polio vaccine (46). Later, hepatitis A vaccine was prepared by Provost and coworkers, also based on chemical inactivation (47). The excellent efficacy of the latter testifies to the ability of careful inactivation to maintain immunogenicity.
Whole inactivated viruses or subunits of virus have been used to make successful vaccines against Japanese encephalitis virus and tick-borne encephalitis virus (48–50).
- Early in the history of bacteriology, morphological studies and chemical analysis showed that many pathogens were surrounded by a polysaccharide capsule and that antibodies against the capsule could promote phagocytosis. The first use of this information to make a vaccine was the development of meningococcal polysaccharide vaccine by Artenstein, Gottschlich, and coworkers (51). This vaccine controlled epidemic and endemic disease in military recruits. Basic bacteriology also suggested that pneumococcal polysaccharides were immunogenic although there were chemical differences between the multiple serotypes. Heidelberg and Macleod and later Austrian fostered the creation of combinations of multiple pneumococcal polysaccharides to prevent invasive infections (52, 53). This principle was then applied to Hemophilus influenzae type b capsular polysaccharide by Anderson, Smith, Schneerson, Robbins, and coworkers (54, 55). The Vi antigen present in the capsule of the typhoid bacillus was made into a vaccine by Landy and coworkers (56).
All of the capsular polysaccharide vaccines generated serum antibodies that prevented bacteremia and thus end-organ disease in adults, but they were not immunogenic in infants, who are unable to mount a B-cell response to polysaccharide alone. This problem was solved by coupling the polysaccharides to proteins, which allowed T-cell help to B cells. In addition, whereas the polysaccharide vaccines did not prevent nasopharyngeal carriage of the bacilli, conjugated vaccines did prevent carriage and thus added the dimension of herd immunity to immunization against the three major bacterial pathogens of infancy. Curiously, the utility of protein conjugation of polysaccharides had been shown by Avery and Goebel in 1929 (57), but this discovery was not taken advantage of until Schneerson, Robbins, and coworkers made a conjugated H. influenzae type b vaccine (55). Eventually, this principle was applied to meningococcal and pneumococcal vaccines, with resulting control of both invasive infections and spread of the organisms. Hib and some meningococcal serogroups have been completely controlled whereas pneumococcal serogroups in vaccines have greatly diminished disease causation.
- Aside from tetanus and diphtheria toxoids, mentioned above under Inactivation, several vaccines consist of partly or fully purified proteins. Most inactivated influenza vaccines used today are generated by growing the viruses in embryonated eggs and then breaking up the whole virus with detergents. The viral hemagglutinin (HA) protein is purified to serve as the vaccine antigen although other components of the influenza virus may be present in the final product (58).
Acellular pertussis vaccines have replaced whole-cell pertussis vaccines in many countries to reduce reactions to the latter. The licensed acellular vaccines consist of one to five proteins from the pertussis bacillus, which are meant to reconstitute efficacy of the whole-cell vaccine without generating febrile reactions. Sato and Sato created the first such vaccine for use in Japan in 1981 (59), but many other acellular vaccines were licensed after extensive trials conducted in the 1990s (60).
Although Pasteur and coworkers made inactivated whole-cell anthrax vaccine early in the history of vaccinology, it was only in the early 1960s that a vaccine was developed for biodefense by the US Army, based on anthrax protective antigen protein secreted by the organism (61). Another improvement on a vaccine originally developed by Pasteur was the creation of a cell culture-produced rabies vaccine by Wiktor, Koprowski, and coworkers in the 1970s (62). Human, monkey, or chicken cells are used to grow the virus, which is then purified and inactivated. The rabies glycoprotein is the protective antigen in the vaccine.
- The revolution of genetic engineering toward the end of the 20th century has greatly impacted vaccine development. The first fruit of that revolution was the vaccine against hepatitis B. Initially, Hilleman and coworkers had purified the hepatitis B surface antigen particles from the serum of naturally infected patients and inactivated any residual live virus (63). However, this type of vaccine could not be practical in the long term. Valenzuela et al. (64) placed the coding sequence for the S antigen into yeast cells and were able to produce large quantities of surface-antigen particles in vitro. Genetic engineering has been used to produce many candidate antigens for vaccines in yeast, animal cells, or insect cells producing an antigen in culture.
Two bacterial live-virus vaccines are administered orally: the Ty21a vaccine against typhoid, which is a strain mutated chemically to deprive the organism of enzymes that contribute to virulence (65), and the CVD103-HgR cholera vaccine, which is unable to synthesize complete cholera toxin (66). Both of these vaccines were made possible after genetic engineering provided the tools for excision of bacterial DNA.
- Many viruses and bacteria are under active study as vectors for vaccine antigens. Poxviruses, adenoviruses, bacillus Calmette–Guérin, and other relatively attenuated microbes have had genes for protective antigens from pathogens inserted into their genomes. The vectors are then injected and undergo either abortive or complete replication, expressing the inserted genes in both cases. The first licensed vector is the 17D yellow fever attenuated strain, which serves as a vector for the prM and E genes of Japanese encephalitis virus, thus immunizing against the latter (67).
The development of the human papilloma virus (HPV) vaccine was made possible because of the properties of the L1 protein of the virus (68, 69). This protein induces protective antibodies, but what makes it particularly immunogenic is that it aggregates to form virus-like particles (VLPs) that are much more immunogenic than the soluble protein. L1 is produced in yeast or insect cells, and the VLPs produced therein form the basis of the current vaccines.
Influenza HA has been produced in insect cells and induces antibodies without the risk of allergy to egg proteins (70, 71).
A vaccine against Lyme disease was on the market briefly. The vaccine consisted of the OspA protein of Borrelia burgdorferi, produced in Escherichia coli (72, 73).
Most recently, a meningococcal group B vaccine has been licensed, consisting of four proteins identified by genomic analysis that induce bactericidal antibodies together with an outer membrane vesicle of the organism (74). This is the first vaccine developed by so-called reverse vaccinology, pioneered by Rappuoli and coworkers (75), by which genomic analysis enables selection of proteins that induce protective immune responses.
- Many have pointed out that it is easier to foretell the past than the future! Be that as it may, the current tendencies in vaccine development are reasonably clear. Although the older methods described above continue to be used, as for example inactivation of whole virus to make vaccines against enterovirus 71 (76), expression of proteins by transcription and translation from either DNA or RNA coding for those proteins will be a widely used approach (77, 78). Attenuated viral or bacterial vectors carrying genetic information for a foreign vaccine antigen is a prominent strategy, exemplified by candidate HIV and dengue vaccines (79, 80). As described above, replicating organisms often make good vaccines, but ways are available to allow only one replication cycle to produce so-called replication-defective agents that maximize safety (81). To generate higher immune responses, stronger adjuvants than aluminum salts are coming into use, including oil-in-water preparations and Toll-like receptor agonists, and their use will surely increase (82).
Meanwhile, structural biology and systems biology are enabling us to identify critical protective antigens and the immune responses they generate, including those that are innate (83, 84). Major unsolved problems remain, including how to deal with immaturity and postmaturity of immune responses in the young and old, respectively; how to induce mucosal responses with nonliving antigens; how to prolong immune memory; and genetic variability as it affects both the safety and efficacy of vaccines. Future vaccines are likely to have a more complex composition than heretofore, but the principles elucidated by past successes will have continued importance as vaccination is extended to more diseases and to all age groups.
“Inactivated Viral Vaccines” (Nov 28, 2014)Edit
Barbara Sanders, Martin Koldijk, and Hanneke Schuitemaker, “Inactivated Viral Vaccines”, Vaccine Analysis: Strategies, Principles, and Control. 2014 Nov 28 : 45–80.
- Inactivated vaccines have been used for over a century to induce protection against viral pathogens. This established approach of vaccine production is relatively straightforward to achieve and there is an augmented safety profile as compared to their live counterparts. Today, there are six viral pathogens for which licensed inactivated vaccines are available with many more in development. Here, we describe the principles of viral inactivation and the application of these principles to vaccine development. Specifically emphasized are the manufacturing procedure and the accompanying assays, of which assays used for monitoring the inactivation process and preservation of neutralizing epitopes, are pivotal. Novel inactivated vaccines in development and the hurdles they face for licensure are also discussed as well as the (dis)advantages of inactivation over the other vaccine production methodologies.
- The first report of “virus” inactivation for vaccine purposes was described in 1886 when Daniel Elmer Salmon and Theobald Smith immunized pigeons with what they thought was a heat-killed hog cholera “virus” (Salmon and Smith 1886). Although in reality it was a cholera-like bacterium, it seeded the scientific community with evidence that immunization with inactivated pathogens can provide protection against infectious disease. Research continued for at least 15 years when at the beginning of the twentieth century the first killed (bacterial) vaccines for humans were developed for typhoid fever, cholera, and plague (Wright and Semple 1897; Haffkine 1899). The foundations of immunization with inactivated virus preparations were also laid at the end of the nineteenth century with Pasteur’s partially inactivated rabies virus (Pasteur et al. 1885), which was cultured in rabbit spinal cords. However, inactivated viral vaccine development was only truly launched with the discovery of cell culture procedures that supported the replication of viral pathogens in vitro, outside the host organism, thus allowing the large scale production of viruses as a source for whole inactivated vaccines. This breakthrough was attributed to Enders, Weller, and Robbins who received the Nobel Prize in 1954 for their discovery on how to cultivate poliovirus in fibroblasts in vitro (Enders et al. 1949; Weller et al. 1949).
In general, all inactivated viral vaccines follow a similar production course in which the pathogen is first cultivated on a substrate to produce large quantities of antigen. Historically, vaccine manufacturers have been using primary cells, tissues, fertilized eggs, and even whole organisms as substrates for virus propagation (Hess et al. 2012; Barrett et al. 2009). Today, vaccine manufacturers are increasingly shifting toward virus growth on continuous cell lines. This brings certain advantages such as reduced production costs, increased vaccine safety, and relatively straightforward upscaling (Barrett et al. 2009). Once the virus has been propagated, it is often purified and concentrated prior to inactivation. Inactivation can be performed using chemical or physical methods or a combination of the two. A wide range of well-established and novel inactivation agents or methods have been described to successfully inactivate viruses for vaccine purposes. Examples are ascorbic acid (Madhusudana et al. 2004), ethylenimine derivatives (Larghi and Nebel 1980), psorlens (Maves et al. 2011), hydrogen peroxide (Amanna et al. 2012), gamma irradiation (Martin et al. 2010a; Alsharifi and Mullbacher 2010), UV treatment (Budowsky et al. 1981), heat (Nims and Plavsic 2012), and many more (Stauffer et al. 2006). Nonetheless, only formaldehyde and β-Propiolactone (BPL) are widely used for inactivation of licensed human viral vaccines for decades.
- Not only do inactivated vaccines possess a higher safety profile as compared to live vaccines, they are also generally less reactogenic, relatively straightforward, and technically feasible to produce with fewer regulatory hurdles for licensure (Zepp 2011). However, inactivated vaccines are typically associated with a lower immunogenicity which can imply the necessity of multiple doses or adjuvant addition which consequently raises the costs of goods and vaccine pricing. Therefore, choosing an inactivated vaccine approach is in general a trade-off with on one hand increased safety (if inactivation is of course complete) and a fast pathway to regulatory approval, but on the other hand the risk of reduced antigenicity of the immunogen which often requires adjuvant addition and/or multiple doses which not only raises production costs but also the complexity of formulation and administration.
- The first reports of vaccination against influenza stem from the 1930s (Stokes et al. 1937) which ultimately lead to the licensure of the first inactivated influenza vaccine in 1945 in the US (Francis et al. 1946; Salk and Francis 1946). Over the course of more than 80 years, the currently available inactivated influenza vaccines have undergone several improvements and have shown significant benefits for society (Clover et al. 1991; Edwards et al. 1994; Gruber et al. 1990; Neuzil et al. 2001; Wilde et al. 1999), however, breadth of protection and efficacy of currently available vaccines are still insufficient to diminish the current annual health burden induced by the virus. Differences in protective efficacy may result from continuing antigenic variation in the prevalent epidemic strains. Due to this variation, the composition of inactivated influenza virus vaccine, unlike that of most viral vaccines, must be kept constantly under review. Accordingly, WHO publishes recommendations concerning the strains to be included in the vaccine twice annually (WHO 2000, 2009a; Ghendon 1991).
Until recently inactivated influenza vaccines consisted of three inactivated viruses; two Influenza A strains and one B strain, however, a new pattern of influenza B circulation has rendered it troublesome to predict the global dominance of one of the two influenza B lineages (Paiva et al. 2013). Therefore, quadrivalent influenza vaccines have been developed to ensure broader protection against Type B influenza viruses as compared to the trivalent vaccines which contained only one Type B influenza strain from one lineage. The licensed quadrivalent inactivated influenza vaccines are formulated in the same way as their trivalent counterparts, however, two influenza B strains, one from the Victoria lineage and one from the Yamagata lineage, are included in the formulation.
After inactivation the vaccine strains are either formulated as virosomes (Herzog et al. 2009), whole inactivated virus (WIV), or detergent-treated “split” vaccines, where the viral envelope is disrupted after inactivation (Wood 1998; Schultz-Cherry and Jones 2010). All the US-licensed inactivated influenza vaccines are split vaccines as “splitting” of the virus is thought to reduce reactogenicity, especially in children (Verma et al. 2012; Nicholson et al. 2003). However, WIV vaccines have been reported to induce stronger immune responses in immunologically naive individuals than split-virus or subunit vaccines (Beyer et al. 1998; Nicholson et al. 1979). Budimir et al. have recently shown that only WIV influenza vaccines, and not split or subunit vaccines, are capable of inducing cross-protection against heterosubtypic challenge due to elicitation of a strong CTL response in mice as whole (BPL) inactivated vaccines are capable of endosomal fusion into the cell cytoplasm (Budimir et al. 2012). The necessity of an influenza vaccine that can elicit cell-mediated immunity and the superiority of WIV vaccines over split vaccine variants has recently been reviewed (Furuya 2012).
- The second currently licensed BPL-inactivated viral vaccine is a rabies vaccine which has an equally rich history of development. Pasteur introduced an experimental rabies vaccine in 1885 when he observed the rapid decrease of rabies virus virulence upon air drying of rabies-infected rabbit spinal cords. Serially less dried rabies-infected rabbit spinal cords containing inactivated—or at least partially inactivated—rabies viruses induced protection of dogs and later humans against challenge following inoculation (Bazin 2011; Pasteur et al. 1885). This method of vaccination, although it was considered a treatment for infected people at the time, was the foundation for rabies vaccines. However, Pasteur faced significant criticism from the scientific community as recipients were essentially inoculated with virulent virus at the end of the treatment (Burke 1996; Gelfand 2002; Wu et al. 2011). This set the incentive to chemically inactivate the rabies virus with phenol in 1908 leading to the first completely inactivated rabies vaccine, despite the disruptive action of phenol on the antigenic sites on the proteins (Fermi 1908; Semple 1911; Briggs 2012).
- In the 1950s and 1960s the vaccine was further improved by using alternative substances to cultivate rabies virus, such as chicken and duck embryos (Peck et al. 1955). This due to the fact that vaccines based on adult mammalian nerve tissue were associated with effects such as encephalomyelitis and demyelination lesions in the CNS due to the presence of myelin (Bonito et al. 2004; Bahri et al. 1996). Therefore, the WHO currently does not recommend the use and production of nerve tissue vaccines (WHO 2005) and has been advocating use of cell culture or embryonated eggs as production platforms since 1983 (WHO 1984). In the US, only cell culture derived rabies vaccines are approved for commercial use, however, some African and Latin American countries continue to produce and use nerve tissue vaccines by phenol inactivation, where the vaccine production protocol resembles the methods from a century ago (Briggs 2012). Today, there are two primary avian cell lines used for rabies vaccine production; purified chick embryo cell vaccine (PCECV) and purified duck embryo rabies vaccine (PDEV) and multiple continuous cell lines such as MRC-5, Vero, and primary hamster kidney cells. However, inactivated vaccines produced on continuous cell lines are not completely free from adverse reactions. There are reports on reactogenicity in response to vaccination with the human diploid cell rabies vaccine (HDCRV) which may relate to the presence of BPL-altered human albumin, added as a stabilizer to vaccine preparations (Anderson et al. 1987; Swanson et al. 1987). Nonetheless, cell culture based vaccines are still vastly preferred over nerve tissue vaccines. Moreover, an additional advantage of the use of a cell line platform, for instance Vero cells, is that they can be cultured in large scale in fermenters on microcarriers which contributes to standardization, safety, and upscaling of the production system resulting in constant yields.
Despite the variation in vaccine cell substrates, the majority of the rabies vaccines are inactivated in a similar manner using a concentration of not more than 1:3,500 and up to 1:5,000 v/v of BPL at 2–8 °C for 24 h (WHO 2007a; Ph. Eur. 2011e). However, there are exceptions such as the use of formalin for Primary Hamster kidney cell culture vaccine (PHKCV). As with the formalin-inactivated vaccines, the inactivation curves have to be validated and approved by the regulatory body. After inactivation, different purification standards can be used such as ultrafiltration, ultracentrifugation, zonal centrifugation, or chromatography. Once formulated, the vaccine potency for all these vaccines is determined by quantifying the degrees of protection against rabies following immunizing and intracerebral challenge of mice (de Moura et al. 2009; Fitzgerald et al. 1978). Based on the results of this National Institutes of Health (NIH) test, the vaccine dosing is set at 2.5 International Units/dose. Many regulatory authorities, including the Ph. Eur. and WHO, have adopted the NIH potency test as the only assay for potency quantification of inactivated Rabies vaccines, despite the recognition of the fact that the animal test should be replaced by an antigen quantification procedure (Bruckner et al. 2003). The vaccine is further tested for complete inactivation by inoculating the cell substrate used for manufacturing with 25 human vaccine doses or more. Cultures are examined for the presence of newly produced rabies virus using immunofluorescence.
- The century old concept of the use of inactivating viruses to elicit protection against the virulent pathogen continues to bear fruit for humanity. Countless improvements and innovations in the field of vaccinology, such as the introduction of recombinant, DNA-based, and vectored vaccines have not stopped the use and development of inactivated vaccines. The relative straightforwardness in which an inactivated vaccine is produced and licensed, accompanied by the fact that inactivated vaccines cannot revert as their replication competent counterparts can do, explains the fact that there are new inactivated pathogens that are being evaluated as vaccine candidates. However, inactivation does not always guarantee the creation of a suitable vaccine as was observed with pathogens such as RSV and measles, therefore immunogenicity of the novel inactivated particle must always be thoroughly tested. Furthermore, new inactivation methods are also being investigated to circumvent the disadvantages of formalin and BPL such as altered immunogenicity due to epitope masking. This section will provide an overview of novel inactivated vaccines in development as well as new inactivation methods.
- The increased safety associated with inactivated vaccines does not entail a spotless track record, as was described for the formalin-inactivated RSV and measles vaccines. The inadequate immune response induced with inactivated viruses is thought to be due to masking of essential epitopes. This drives the investigation of alternative inactivation methods that do not alter epitopes or skew immune responses to ensure a protective vaccine with high efficiency. Three new inactivation methods, being hydrogen peroxide treatment, zinc-finger reactive treatment, and gamma irradiation are described in more detail below. Whether these inactivation methods will be implemented in the manufacturing of vaccines remains to be determined.
- The oral polio vaccine (OPV) displays frequent reversion to virulence in vaccine recipients and there are estimates of approximately 400–800 vaccine-associated paralytic poliomyelitis (VAPP) cases per year globally (John 2002). Despite the immediate recognition of the fact that OPV strains can revert readily into a pathogenic phenotype (Henderson et al. 1964), OPV has been used since the 1960s and still is being used extensively. However, recently, it has been acknowledged that use of the oral live attenuated vaccine is at odds with global eradication of poliomyelitis. Indeed, the number of vaccine-associated poliomyelitis cases is in the range of wild-type PV induced poliomyelitis cases (WHO 2006a). Although IPV is a safe alternative, the costs of currently available IPV are too high to implement its use in low income countries (Heinsbroek and Ruitenberg 2010; Zehrung 2010) and several options to reduce costs of IPV are being considered (WHO 2009b). In the era after eradication, IPV use will have to be continued at least for a certain amount of time. At that time, production of IPV from wild-type PV strains will fall under strict biosafety measures (WHO 2009c). Even though it is currently not clear whether an IPV based on OPV strains may be produced at lower biosafety level after eradication as compared to a wild-type based IPV, there is much research going on to the inactivation of the OPV strains with formalin to eventually replace the inactivated PV vaccine based on the wild-type strains. Not only would the lowering of biosafety level decrease potential costs of goods, replacing the wild-type strains greatly reduces the risks of poliomyelitis upon accidental outbreaks from the manufacturing facility, after eradication. The manufacture of Sabin-IPV is essentially identical to the Salk-IPV process with slight modifications (Westdijk et al. 2011). The WHO encourages the development of this Sabin-IPV vaccine (Bakker et al. 2011) and multiple clinical trials have been or are being performed (Verdijk et al. 2011), moreover, in Japan a Sabin based IPV has recently been licensed in combination with diphtheria, tetanus, and acellular pertussis (DTaP-Sabin IPV) (Mahmood et al. 2013). In general, Sabin-IPV displays higher immunogenicity for serotype 1, lower for type 2, and similar for type 3 in comparison to Salk-IPV, licensure of more Sabin derived IPV’s is foreseen in the near future.
Monath et al. describe the results of a Phase I study of a BPL-inactivated Yellow Fever (YF) vaccine, based on the licensed attenuated 17D strain (Monath et al. 2011). The 17D vaccine was developed in 1936 by Max Theiler and today 20 million doses are issued per year. However, yellow fever vaccine-associated viscerotropic disease (YF-AVD) and yellow fever vaccine-associated neurological disease (YF-AND) occurring at a frequency of 0.4 and 1.8 per 100,000 doses, respectively (Lindsey et al. 2008), instigate a need for safer vaccines. Inactivated vaccines will reduce the adverse effects associated with the vaccine and is predicted to be less reactogenic as it has been cultivated on Vero cells instead of eggs (Hayes 2010). The alum-adjuvanted, BPL-inactivated vaccine induced neutralizing antibodies in a high percentage of subjects, albeit lower titers than the live vaccine, whether the lower titers will be compensated for by the higher safety profile is yet to be determined (Monath et al. 2011).
“Advancements in DNA vaccine vectors, non-mechanical delivery methods, and molecular adjuvants to increase immunogenicity” (Dec 2, 2017)Edit
John J Suschak, James A Williams, Connie S Schmaljohn; “Advancements in DNA vaccine vectors, non-mechanical delivery methods, and molecular adjuvants to increase immunogenicity”, Hum Vaccin Immunother, 2017 Dec 2;13(12):2837-2848.
- A major advantage of DNA vaccination is the ability to induce both humoral and cellular immune responses. DNA vaccines are currently used in veterinary medicine, but have not achieved widespread acceptance for use in humans due to their low immunogenicity in early clinical studies. However, recent clinical data have re-established the value of DNA vaccines, particularly in priming high-level antigen-specific antibody responses. Several approaches have been investigated for improving DNA vaccine efficacy, including advancements in DNA vaccine vector design, the inclusion of genetically engineered cytokine adjuvants, and novel non-mechanical delivery methods. These strategies have shown promise, resulting in augmented adaptive immune responses in not only mice, but also in large animal models. Here, we review advancements in each of these areas that show promise for increasing the immunogenicity of DNA vaccines.
- The constant emergence, and re-emergence, of known and novel pathogens challenges researchers to develop new vaccination technologies that allow for the rapid development of safe and effective vaccines. Nucleic acid (DNA and RNA) vaccines have characteristics that meet these challenges, including ease of production, scalability, consistency between lots, storage, and safety. DNA vaccine technology usually is based on bacterial plasmids that encode the polypeptide sequence of candidate antigens. The encoded antigen is expressed under a strong eukaryotic promoter, yielding high levels of transgene expression.1 Inclusion of transcriptional enhancers, such as Intron A, enhance the rate of polyadenylation and nuclear transport of messenger RNA (mRNA).2 The vaccine plasmids are generally produced in bacterial culture, purified, and then used to inoculate the host.
Modern DNA vaccine design generally relies on synthesis of the nucleic acid and possibly one-step cloning into the plasmid vector, reducing both the cost and the time to manufacture. Plasmid DNA is also extremely stable at room temperature, reducing the need for a cold chain during transportation. Vaccination with DNA plasmid removes the necessity for protein purification from infectious pathogens, improving safety. Furthermore, DNA vaccination has an excellent safety profile in the clinic, with the most common side effect being mild inflammation at the injection site.3 Importantly, DNA vaccines provide a safe, non-live vaccine approach to inducing balanced immune responses, as the in vivo production of antigen allows for presentation on both class I and class II major histocompatibility complex (MHC) molecules (Fig. 1). This elicits antigen specific antibodies,4 as well as cytotoxic T lymphocyte responses (CTL),5 something that remains elusive in most non-live vaccines. DNA vaccines have also demonstrated the ability to generate follicular T helper populations,6 which are critical for the induction of high quality antigen-specific B cell responses.7
- DNA vaccination has proven successful in several animal models for preventing or treating infectious diseases, allergies, cancer, and autoimmunity.8-12 The early success of small animal studies led to several human clinical trials. However, the protective immunity observed in small animals and non-human primates was not observed in human studies when DNA vaccines were administered alone by needle delivery. Like the more conventional protein-based vaccines, DNA can be delivered by a variety of routes, including intramuscular (IM), intradermal (ID), mucosal, or transdermal delivery. Because DNA plasmids must enter host cell nuclei to be transcribed into mRNA, the early failure of DNA vaccines to elicit strong responses in humans was largely due to their delivery by needle injection, which deposits the DNA in intracellular spaces, rather than within cells. Improved delivery technologies, such as intramuscular or intradermal electroporation, have been used to facilitate transport of DNA into cells, resulting in much better immunogenicity in both clinical and non-clinical studies.13-19 In one study, electroporation-enhanced DNA vaccination resulted in increased polyfunctional antigen-specific CD8+ T cells in patients receiving a HPV DNA vaccine expressing the E6 and E7 genes of HPV16 and HPV18 respectively.20 The majority of DNA vaccinated patients displayed complete regression of their cervical lesions, as well as viral clearance, following DNA delivery. Other mechanical delivery approaches use physical force such as particle bombardment (gene gun) to deliver the DNA plasmids into targeted tissues or cells, with some clinical successes.21-23 Delivery of a Hepatitis B DNA vaccine by particle bombardment resulted in sustained antibody titers in subjects who had previously failed to respond to a licensed subunit vaccine.23 Needle-free pneumatic or jet injectors have also shown promise in both animal and human clinical trials,24-27 and function by injecting a high-pressure, narrow stream of injection liquid into the epidermis or muscles of test subjects. In addition to these improved mechanical delivery methods, several other approaches are being explored to increase the immunogenicity of DNA vaccines in humans. Here we review 3 of these approaches which show promise for advancing DNA vaccines: non-mechanical delivery, inclusion of molecular adjuvants, and improvements in DNA vaccine vectors.
- As already mentioned, the greatest impediment to DNA vaccination is low immunogenicity due to difficulties in delivering DNA plasmid into the host cell. The transportation of DNA vaccine plasmids into cellular nuclei requires the crossing of several barriers. Vaccine plasmid must cross the phospholipid cellular membrane through endocytosis or pinocytosis, escape degradation in endosomes and lysosomes, survive cytosolic nucleases, and translocate across the nuclear envelope. In contrast to physical delivery systems, chemical delivery approaches use biopharmaceuticals to increase DNA vaccine transfection efficiency.
- The use of liposomes as a carrier molecule has become a popular DNA vaccine delivery method as liposomes not only enhance transfection efficiency, but also have an adjuvant effect. Liposomes are spherical vesicles composed of phospholipids and cholesterol arranged into a lipid bilayer, allowing for fusion with cellular lipid membranes.28 DNA plasmid can be either bound to the liposome surface, or encased within the hydrophobic core of the liposome. This facilitates delivery of the DNA vaccine plasmid into the cells. Importantly, lipid vesicles can be formulated as either unilamellar or multilamellar. Multilamellar vesicles allow for sustained delivery of vaccine over an extended period of time. While the use of liposomes for IM injection has resulted in some reactogenicity issues,29,30 liposome/DNA vaccine complexes have demonstrated an immunological benefit. IM injection of a liposome/influenza nucleoprotein formulation increased antibody titers 20-fold compared with vaccine alone.31,32 Boosting of antibody titers did not diminish the cytotoxic T cell response. Likewise, inclusion of a liposome formulation in a P. falciparum vaccine enhanced the IFN-γ production.33,34 An ensuing human trial involving DNA plasmids encoding the influenza H5 HA, nucleoprotein, and M2 genes reported cellular immune response rates and antibody titers comparable to that of the currently available inactivated protein-based H5 vaccines.35 Additionally, liposomes have shown promise as a candidate for delivery of DNA vaccines to mucosal tissue.36 A recent study demonstrated that vaccination with liposome encapsulated influenza A virus M1 induced both humoral and cellular immune responses that protected against respiratory infection.36 Liposomes have also been shown to be an effective delivery method for intranasal DNA vaccination, conferring protective immune responses against infection.37,38
- DNA vaccine delivery can also be accomplished through the use of biodegradable polymeric micro- and nanoparticles consisting of amphiphilic molecules between 0.5–10 µm in size. Similar to loading of DNA plasmid on liposomes, plasmid molecules can be either encapsulated or adsorbed onto the surface of the nanoparticles.39-42 These particles function as a carrier system, protecting the vaccine plasmid from degradation by extracellular deoxyribonucleases. In addition to shielding plasmid DNA from nucleases, micro- and nanoparticles promote the sustained release of vaccine instead of the bolus type of delivery characteristic of larger submicrometer complexes.39,43 High molecular weight cationic polymers have proven significantly more effective than cationic liposomes in aggregating DNA vaccine plasmid. Plasmid DNA immobilized within biodegradable chitosan-coated polymeric microspheres (ranging from 20 to 500 μm) can induce both mucosal and systemic immune responses.44 Microspheres may be delivered either by the oral or intraperitoneal route, allowing for direct transfection of dendritic cells (DC), thereby increasing DC activation. The benefits of microsphere formulations have been shown in mice, non-human primates, and humans 45-49 against a wide range of diseases including hepatitis B,50 tuberculosis,51 and cancer.52 These results suggest that microparticle-based delivery systems are capable of significantly improving DNA vaccine immunogenicity, and boosting cellular and humoral immune responses.
The use of liposomes or nanoparticles appears to be safe and well tolerated in clinical studies. Microparticle-based delivery systems can increase gene expression, as well as, DNA vaccine immunogenicity. Although many of the earliest carrier formulations did not show a significant clinical benefit, more recent studies highlighted herein yielded promising clinical data. As microparticles can be prepared with significant structural diversity (size, surface charge, lipid content), they offer considerable flexibility of vaccine formulation. This allows for optimization of the vaccine based on the specific needs of the clinician.
- A major advantage of DNA vaccination is the ability of multiple molecules such as molecular adjuvants to be inserted into the plasmid. Unlike the addition of recombinant cytokines, co-stimulatory molecules, and TLR ligands, which have a limited duration due to the short half-life of recombinant protein in vivo, molecular adjuvant-encoding plasmids will express protein for the same duration as the antigen, stimulating the immune system for a greater length of time. This can be done without fear of eliciting a cytokine storm, as generation of the adjuvanting signal will be localized to the site of vaccination. Of note, homologous recombination between plasmid-encoded cytokines and the host gene sequence does not appear to be a significant concern, as multiple studies have shown that only extrachromosomal plasmid DNA has been identified following intramuscular injection.121,122 Furthermore, many current plasmids have been-codon optimized to improve gene expression in mammalian cells. This has resulted in changes to the cytokine gene sequence, limiting the possibility for homologous recombination and/or integration. Molecular adjuvants therefore show great promise for both increasing immunogenicity and extending the longevity of the immune response.
- Another approach to improve DNA vaccines is to engineer the vector to increase innate immune activation. DNA vaccines are potent triggers of innate immunity. Various studies have determined several innate immune pathways are activated by DNA vaccination (Fig. 2). Most of the intrinsic adjuvant effect of DNA is mediated by cytoplasmic innate immune receptors that nonspecifically recognize B DNA and activate Sting or Inflammasome mediated signaling,53,143 but unmethylated CpG sequences specific for TLR9 activation may also be important for priming CD8 T cell responses.144,145 Along these lines, DNA vaccine vectors may be sequence modified to introduce immunostimulatory xxCGxx TLR9 agonists into the vector to increase innate immune activation. This approach has been used to improve DNA vaccine immunogenicity,58,59,146 but the results are variable. Some of the variability may be due to unintended inhibition of the eukaryotic promoter expression resulting from integration of CpG motifs into non-permissive sites in the vector.125 As well, certain DNA delivery methods may not transfer DNA to the endosome as effectively as other deliveries (e.g. liposomes), preventing unmethylated CpG interaction with, and activation of, TLR9. Part of the complexity is that optimal TLR9 activating xxCGxx motifs are species-specific; different xxCGxx agonist motifs differentially modulate the immune response147 and many xxCGxx motifs are immunosuppressive.
- DNA vaccines encoding immunostimulatory sequences that selectively improve CTL responses to encoded antigen may have niche application in vaccines for intracellular pathogens or cancer. Innovations that increase transgene expression may be used to improve the performance of immunomodulatory molecular adjuvant plasmids, in addition to traditional antigen expressing DNA vaccine plasmids. Collectively, vector design innovations that improve transgene expression level and innate immune activation are complementary to improved mechanical and non-mechanical DNA vaccine delivery platforms. Combining improved vectors with liposome or polymeric particle non-mechanical delivery, or with needle free injector device delivery, has the potential to increase immunogenicity with these well tolerated, safe, delivery platforms.
- While DNA vaccination provides several advantages over more conventional vaccination strategies, further optimization is necessary before it becomes the predominant strategy in human patients. Despite initial setbacks, significant progress has been made in overcoming the problem of low immunogenicity in humans. A clearer understanding of the immune mechanisms governing DNA vaccine immunogenicity has illuminated several pathways that may be useful in further improving DNA vaccine efficacy. A large catalog of cytokines, chemokines, adhesion molecules, and transcription factors are in the process of being tested as molecular adjuvants, although it is likely that each will need to be carefully assessed for safety and tolerability. Likewise, continued development of vaccine delivery methods appears promising. New formulations exploiting sustained vaccine delivery methods, such as slow-releasing micropatches or multilamellar vesicles, are on the horizon. The strong appeal of needle-free injection and mucosal delivery, the ease of design, and the recent clinical successes with DNA vaccines suggests that this approach is on the precipice of redefining the field of vaccinology.